Anti-CD74 Immunoconjugates and Methods of Use

ABSTRACT

Disclosed are compositions that include anti-CD74 immunoconjugates and optionally a therapeutic and/or diagnostic agent. In preferred embodiments, the immunoconjugates comprise one or more anti-CD74 antibodies or antigen-binding fragments thereof, conjugated to a carrier such as a polymer, nanoparticle, complex or micelle. Also disclosed are methods for preparing the immunoconjugates and using the immunoconjugates in diagnostic and therapeutic procedures. In certain preferred embodiments, the therapeutic methods comprise administering to a subject with a CD74-expressing disease an anti-CD74 immunoconjugate and thereby inducing cell death of CD74-expressing cells. In more preferred embodiments, the CD74 immunoconjugate is capable of inducing cell death in the absence of any other therapeutic agent, although such agents may be optionally administered prior to, together with or subsequent to administration of the anti-CD74 immunoconjugate. The compositions may be part of a kit for administering the anti-CD74 immunoconjugates or compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 12/789,553,filed on May 28, 2010, which was a continuation-in-part of applicationSer. No. 10/706,852, filed on Nov. 12, 2003, now U.S. Pat. No.7,829,0640, which claimed the benefit under 35 U.S.C. 119(e) ofprovisional application Ser. No. 60/478,830, filed on Jun. 17, 2003, andwhich was a continuation-in-part of application Ser. No. 10/314,330,filed on Dec. 9, 2002, now U.S. Pat. No. 7,837,995, which was acontinuation of application Ser. No. 09/965,796, filed on Oct. 1, 2001,now U.S. Pat. No. 7,910,103, which was a continuation of applicationSer. No. 09/307,816, filed on May 10, 1999, now U.S. Pat. No. 6,306,393.U.S. Ser. No. 10/706,852 was also a continuation-in-part of applicationSer. No. 10/350,096, filed on Jan. 24, 2003, which was a continuation ofapplication Ser. No. 09/590,284, filed on Jun. 9, 2000, now U.S. Pat.No. 7,074,403. U.S. Ser. No. 10/706,852 was also a continuation-in-partof application Ser. No. 10/377,122, filed on Mar. 3, 2003, now U.S. Pat.No. 7,312,318, which claimed the benefit of provisional application Ser.No. 60/360,259, filed on Mar. 1, 2002.

This application is also a continuation-in-part of application Ser. No.12/644,146, filed Dec. 22, 2009, now U.S. Pat. No. 7,981,398, which wasa divisional of application Ser. No. 11/925,408, filed Oct. 26, 2007,now U.S. Pat. No. 7,666,400, which was a continuation-in-part of U.S.Pat. Nos. 7,521,056; 7,527,787; 7,534,866 and 7,550,143; which claimedthe benefit under 35 U.S.C. 119(e) of Provisional U.S. PatentApplication Ser. Nos. 60/668,603, filed Apr. 6, 2005; 60/728,292, filedOct. 19, 2005; 60/751,196, filed Dec. 16, 2005; 60/782,332, filed Mar.14, 2006; and 60/864,530, filed Nov. 6, 2006. All of theabove-identified applications and patents are incorporated herein byreference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jun. 24, 2010, is namedIMM211US.txt, and is 27,665 bytes in size.

FIELD OF THE INVENTION

The present invention concerns compositions and methods of use ofnanoparticles, polymers or other complexes attached to targetingmolecules, such as antibodies, antibody fragments or antibody fusionproteins. More particularly, the antibodies, fragments or fusionproteins may bind to a tumor associated antigen, infectious diseaseassociated antigen or other disease associated antigen. Even moreparticularly, the targeting molecule may bind to the invariant chain(Ii) of the HLA-DR complex, also known as CD74. An exemplary anti-CD74antibody is milatuzumab (hLL1). The antibody conjugated particle orcomplex is of use for the treatment of a variety of diseases, such asautoimmune disease, immune dysregulation disease, cancer, lymphoma,leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffusedlarge B cell lymphoma, multiple myeloma or non-Hodgkin's lymphoma.

BACKGROUND

Liposomes, nanoparticles, and polymers have been used as carriers fortherapeutic agents, where antibodies may be conjugated to the carrier toprovide specific targeting of the carrier/agent complex to a diseasedcell or tissue. (See, e.g., Xu et al., Mol. Cancer. Ther., 1:337-346(2002); Torchilin, et al., Proc. Nat'l. Acad. Sci., 10: 6039 (2003);U.S. Pat. No. 6,165,440; U.S. Pat. No. 5,702,727; U.S. Pat. No.5,620,708; U.S. Pat. No. 5,565,215; U.S. Pat. No. 6,530,944; U.S. Pat.No. 6,562,318; U.S. Pat. No. 6,558,648; and U.S. Pat. No. 6,395,276).More recently, antibodies, antibody fragments and other therapeuticagents have been conjugated to liposomes, nanoparticles, polymers suchas polyethylene glycol (PEG) or other complexes to optimize theirpharmacokinetic or other properties. (See, e.g., U.S. Patent ApplicationPubl. No. 20090060862, filed Oct. 26, 2007, and U.S. Pat. Nos.7,521,056; 7,527,787; 7,534,866 and 7,550,143, the Examples section ofeach of which is incorporated herein by reference.) The antibody orantibodies of choice for targeting purposes may bind to a known tumorassociated antigen (TAA) or to an antigen associated with infectiousdiseases or other disease states.

One such tumor-associated antigen is CD74, which is an epitope of themajor histocompatibility complex (MHC) class II antigen invariant chain,Ii, present on the cell surface and taken up in large amounts of up to8×10⁶ molecules per cell per day (Hansen et al., 1996, Biochem. J., 320:293-300). CD74 is present on the cell surface of B-lymphocytes,monocytes and histocytes, human B-lymphoma cell lines, melanomas, T-celllymphomas and a variety of other tumor cell types. (Hansen et al., 1996,Biochem. J., 320: 293-300) CD74 associates with α/β chain MHC IIheterodimers to form MHC II αβIi complexes that are involved in antigenprocessing and presentation to T cells (Dixon et al., 2006, Biochemistry45:5228-34; Loss et al., 1993, J Immunol 150:3187-97; Cresswell et al.,1996; Cell 84:505-7).

CD74 also plays an important role in cell proliferation and survival.Binding of the CD74 ligand, macrophage migration inhibitory factor(MIF), to CD74 activates the MAP kinase cascade and promotes cellproliferation (Leng et al., 2003, J Exp Med 197:1467-76). Binding of MIFto CD74 also enhances cell survival through activation of NF-κB andBcl-2 (Lantner et al., 2007, Blood 110:4303-11).

Murine LL1 (mLL1 or murine anti-CD74 antibody) is a specific monoclonalantibody (mAb) reactive with CD74. Cell surface-bound LL1 is rapidlyinternalized to the lysosomal compartment and quickly catabolized, muchfaster than other antibodies, such as anti-CD19 or anti-CD22 (Hansen etal., 1996, Biochem. J., 320: 293-300). LL1 was reported to exhibit thehighest rate of accumulation inside B cells of any of the antibodiestested (Griffiths et al., 2003, Clin Cancer Res 9:6567-71).

Murine LL1 was developed by fusion of mouse myeloma cells withsplenocytes from BALB/c mice immunized with preparations from the RajiB-lymphoma cell line (called EPB-1 in Pawlak-Byczkowska et al., Can.Res., 49: 4568 (1989)). The clinical use of mLL1, just as with mostother promising murine antibodies, has been limited by the developmentin humans of a human anti-mouse antibody (HAMA) response. A HAMAresponse is generally not observed following injection of mLL1 Fab′, asevidenced in a bone marrow imaging study using an mLL1 Fab′ labeled with^(99m)Tc. Juweid et al., Nuc. Med. Comm. 18: 142-148 (1997). However, insome therapeutic and diagnostic uses, a full-length anti-CD74 antibodymay be preferred. This use of the full-length anti-CD74 antibody canlimit the diagnostic and therapeutic usefulness of such antibodies andantibody conjugates, not only because of the potential anaphylacticproblem, but also as a major portion of the circulating conjugate may becomplexed to and sequestered by the circulating anti-mouse antibodies.Although the use of antibody fragments of mLL1 may circumvent theproblems of immunogenicity, there are circumstances in which whole IgGis more desirable and the induction of cellular immunity is intended fortherapy or enhanced antibody survival time. In general, HAMA responsespose a potential obstacle to realizing the full diagnostic andtherapeutic potential of murine anti-CD74 antibodies. Therefore, thedevelopment of immunoconjugates that include chimeric, humanized andhuman anti-CD74 binding molecules, (e.g., antibodies and fragmentsthereof, antibody fusion proteins thereof, multivalent and/ormultispecific antibodies and fragments thereof), would be extremelyuseful for therapy and diagnosis, with reduced production of humananti-mouse antibodies.

SUMMARY

Disclosed is a composition that includes an immunoconjugate, where theimmunoconjugate includes an anti-CD74 binding molecule conjugated to oneor more carriers. The carrier may include molecules that can form ahigher-ordered structure, (such as lipids or polymers), or the carriermay be a higher-ordered structure itself, (such as a micelle ornanoparticle). In preferred embodiments, the anti-CD74 binding moleculeis an antibody, antigen-binding antibody fragment, antibody fusionprotein or multispecific antibody or fragment thereof. In certainembodiments, the composition may also comprise one or more additionaleffector molecules, such as a radionuclide, a chemotherapeutic agent, atoxin, an enzyme, an immunomodulator or a second antibody or fragmentthereof. The additional effector(s) may be contained within an emulsionor attached to the anti-CD74 antibody. However, the skilled artisan willrealize that methods of therapy of common disease states, such ascancer, may involve the administration to a subject of two or moredifferent therapeutic agents (effector molecules) concurrently,consecutively or separately.

The anti-CD74 binding molecule may be conjugated or linked to thecarrier by a number of linkages including sulfide linkages, hydrazonelinkages, hydrazine linkages, ester linkages, amido linkages, aminolinkages, imino linkages, thiosemicarbazone linkages, semicarbazonelinkages, oxime linkages, and carbon-carbon linkages. A sulfide linkagemay be preferred, where the binding molecule may include disulfidelinkages, which may be reduced to provide free thiol groups.

The composition may include additional binding molecules, (e.g.,antibodies or fragments thereof that bind to CD19, CD20, CD22, CD30,CD33, CD52, CD80, HLA-DR, MUC1, TAC, IL-6, tenascin, VEGF, placentalgrowth factor, carbonic anhydrase IX, and mixtures thereof). Theadditional binding molecules may be covalently or non-covalentlyassociated with any component of the composition (e.g., the carrier).

Where the carrier is a lipid, preferably the lipid is capable of formingan emulsion or a higher-ordered structure such as a micelle. Forexample, the lipid may be amphiphilic. To facilitate conjugation to theanti-CD74 binding molecule, the lipid may contain one or more groupscapable of reacting with the anti-CD74 binding molecule, such asnucleophilic carbons, (e.g., at a distal terminus). In one embodiment,the lipid is polyethyleneglycol (PEG)-maleimide and the anti-CD74binding molecule reacts via free thiol groups with the maleimide group.Maleimide groups may also be present on other carriers as describedherein for conjugating the anti-CD74 binding molecule. For example,nanoparticles may contain maleimide groups for conjugating the anti-CD74binding molecule. In addition to maleimide groups, other groups forconjugating binding molecules may include vinylsulfones.

The composition may include therapeutic or diagnostic agents, which maybe covalently, non-covalently, or otherwise associated with anycomponent of the composition. For example, the therapeutic or diagnosticagent may be covalently linked to the anti-CD74 binding molecule.Alternatively, the therapeutic or diagnostic agent may be covalentlylinked to the carrier or non-covalently or otherwise associated with thecarrier.

The effector may comprise any number of therapeutic or diagnosticagents. For example, the effector may include a drug, prodrug, toxin,enzyme, radioisotope, immunomodulator, cytokine, hormone, bindingmolecule (e.g., an antibody), or an oligonucleotide molecule (e.g., anantisense molecule or a gene). Antisense molecules may include antisensemolecules that correspond to bcl-2 or p53. The effector may includeaplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin,irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycinglucuronide, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinylestradiol, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel,pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol,testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, velcade, vinblastine, vinorelbine,vincristine, ricin, abrin, ribonuclease, onconase, rapLR1, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin, orcombinations thereof. In certain embodiments, the effector includesFUdR, or FUdR-dO.

The composition may also include one or more hard acid chelators or softacid chelators. For example, the chelator may include NOTA, DOTA, DTPA,TETA, Tscg-Cys, or Tsca-Cys. In certain embodiments, the chelators mayform complexes with cations selected from Group II, Group III, Group IV,Group V, transition, lanthanide or actinide metal cations, or mixturesthereof. Alternatively, the cations may be covalently, non-covalently,or otherwise associated with any component of the complex. In certainembodiments, the composition includes cations selected from Tc, Re, Bi,Cu, As, Ag, Au, At, or Pb.

The composition may also include a nuclide (e.g., a radionuclide). Thenuclide may be selected from a number of nuclides including ⁸F, ³²P,³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Se, ⁷⁷As,⁸⁶Y, ⁸⁹Sr, ⁸⁹Zr, ⁹⁰Y, ⁹⁴Tc, ^(94m)Tc, ⁹⁹Mo, ^(99m)Tc, ¹⁰⁵Pd, ¹⁰⁵Rh,¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm,¹⁵⁴⁻¹⁵⁸Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra, or²²⁵Ac.

Where the effector is an enzyme, suitable enzymes may includecarboxylesterases, glucuronidases, carboxypeptidases, beta-lactamases,phosphatases, and mixtures thereof. Where the effector is animmunomodulator, suitable immunomodulators may include IL-1, IL-2, IL-3,IL-6, IL-10, IL-12, IL-18, IL-21, interferon-α, interferon-β,interferon-γ, G-CSF, GM-CSF, and mixtures thereof. The effector may alsoinclude an anti-angiogenic agent, e.g., angiostatin, endostatin,basculostatin, canstatin, maspin, anti-VEGF binding molecules,anti-placental growth factor binding molecules, or anti-vascular growthfactor binding molecules.

Preferably, the anti-CD74 binding molecule may be LL1 or a fragmentthereof, although any anti-CD74 binding molecule is suitable. Forexample, production of monoclonal antibodies is well known in the art.See Harlow & Lane (eds), Antibodies. A Laboratory Manual, Cold SpringHarbor Laboratory, NY. However, human, chimeric, or humanizedderivatives of LL1 or fragments thereof may be particularly suitable.Derivatives of LL1 are described in U.S. Pat. No. 7,312,318 and U.S.Patent Application Ser. No. 60/360,259, filed Mar. 1, 2002, which areincorporated herein by reference in their entireties. The anti-CD74binding molecule or fragment thereof may be monoclonal.

The anti-CD74 binding molecule may include a human, chimeric, orhumanized anti-CD74 antibody or fragment thereof. For example, a bindingmolecule may contain the CDRs of the light and heavy chain variableregions of a murine anti-CD74 antibody. A humanized anti-CD74 antibodyor fragment may include the complementarity-determining regions (CDRs)of murine anti-CD74 (mLL1) and human antibody constant and framework(FR) region sequences, which may be substituted with at least one aminoacid from corresponding FRs of a murine antibody. An antibody orfragment may include a humanized IgG1.

An anti-CD74 binding molecule may be a chimeric anti-CD74 antibody orfragment thereof and may include the light and heavy chain variableregions of a murine anti-CD74 antibody, attached to human antibodyconstant regions. A chimeric antibody or fragment thereof may include achimeric IgG1 or fragment thereof.

The anti-CD74 binding molecule may be selected such that the binding ofthe molecule or fragment thereof to CD74 competes with or is blocked byan antibody or fragment thereof specific for CD74, such as the LL1antibody. Alternatively, the binding molecule may be selected such thatthe binding molecule binds to the same epitope of CD74 as an antibody orfragment thereof specific for CD74, such as the LL1 antibody. In stillother alternatives, the binding molecule may be selected to beinternalized by Raji lymphoma cells in culture. In another embodiment,an anti-CD74 binding molecule, such as an antibody or fragment thereof,may be selected such that it induces apoptosis of Raji cells in cellculture when cross-linked with goat antisera reactive with the Fc of amurine IgG1 antibody.

The anti-CD74 binding molecule may also include a fragment whichincludes a F(ab′)₂, Fab, scFv, Fv, or a fusion protein utilizing part orall of the light and heavy chains of the F(ab)₂, Fab, scFv, or Fv, suchthat the fragment is capable of binding to CD74. The binding moleculemay be selected or designed to be multivalent, or multivalent andmultispecific. The fragments may form a bispecific binding molecule or adiabody. In one embodiment, the binding molecule includes a fusionprotein that includes four or more Fvs, or Fab's of the antibodies orfragments thereof. In a further embodiment, the binding moleculeincludes a fusion protein that includes one or more Fvs or Fab's of ananti-CD74 antibody or fragment thereof, and one or more Fvs or Fab'sfrom an antibody specific for a tumor cell marker that is not a CD74antigen. For example, the tumor cell marker may include a B-cell lineageantigen such as CD19, CD20, or CD22. Alternatively, the tumor cellmarker may include HLA-DR, CD30, CD33, CD52, MUC1 or TAC.

The anti-CD 74 binding molecule may include human constant regions ofIgG1, IgG2a, IgG3, or IgG4.

In certain preferred embodiments, the anti-CD74 complex may be formed bya technique known as dock-and-lock (DNL) (see, e.g., U.S. Pat. Nos.7,521,056; 7,527,787; 7,534,866; 7,550,143 and U.S. Patent Publ. No.20090060862, filed Oct. 26, 2007, the Examples section of each of whichis incorporated herein by reference.) Generally, the DNL technique takesadvantage of the specific and high-affinity binding interaction betweena dimerization and docking domain (DDD) sequence derived fromcAMP-dependent protein kinase and an anchor domain (AD) sequence derivedfrom any of a variety of AKAP proteins. The DDD and AD peptides may beattached to any protein, peptide or other molecule. Because the DDDsequences spontaneously dimerize and bind to the AD sequence, the DNLtechnique allows the formation of complexes between any selectedmolecules that may be attached to DDD or AD sequences. Although thestandard DNL complex comprises a trimer with two DDD-linked moleculesattached to one AD-linked molecule, variations in complex structureallow the formation of dimers, trimers, tetramers, pentamers, hexamersand other multimers. In some embodiments, the DNL complex may comprisetwo or more anti-CD74 antibodies, antibody fragments or fusion proteinswhich may also be attached to one or more other effectors or polymers,such as PEG.

Also disclosed is a method for treating and/or diagnosing a disease ordisorder that includes administering to a patient a therapeutic and/ordiagnostic composition. The therapeutic and/or diagnostic compositionincludes any of the aforementioned compositions, generally a compositionthat includes: (1) one or more anti-CD74 binding molecules or fragmentsthereof conjugated to a carrier; (2) a pharmaceutically acceptableexcipient; and (3) optionally an effector molecule (e.g., a therapeuticor diagnostic agent). Typically, the composition is administered to thepatient intravenously, intramuscularly or subcutaneously at a dose of20-5000 mg.

In preferred embodiments, the disease or disorder is associated withCD74-expressing cells and may be a cancer, an immune dysregulationdisease, an autoimmune disease, an organ-graft rejection, agraft-versus-host disease, a solid tumor, non-Hodgkin's lymphoma,Hodgkin's lymphoma, multiple myeloma, a B-cell malignancy, or a T-cellmalignancy. A B-cell malignancy may-include indolent forms of B-celllymphomas, aggressive forms of B-cell lymphomas, chronic lymphaticleukemias, acute lymphatic leukemias, and/or multiple myeloma. Solidtumors may include melanomas, carcinomas, sarcomas, and/or gliomas. Acarcinoma may include renal carcinoma, lung carcinoma, intestinalcarcinoma, stomach carcinoma, breast carcinoma, prostate cancer, ovariancancer, and/or melanoma.

In one embodiment, the composition may comprise an agent forphotodynamic therapy, e.g., a photosensitizer such as a benzoporphyrinmonoacid ring A (BDP-MA), tin etiopurpurin (SnET2), sulfonated aluminumphthalocyanine (AISPc), or lutetium texaphyrin (Lutex). The method mayalso include administering an irradiating light source to the targetedcells or tissue. In certain embodiments, photodynamic therapy may beused diagnostically as well as therapeutically.

The method may include administering a composition that includes adiagnostic nuclide, e.g., ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y,⁸⁹Zr, ⁹⁴Tc, ^(94m)Tc, ^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I.Typically, the diagnostic nuclide will emit 25-4000 keV gamma particlesand/or positrons.

The method may include administering a composition that includes adiagnostic agent, which can be used to perform positron emissiontomography (PET), such as ¹⁸F or ⁶⁸Ga. As such, the method may includeperforming positron-emission tomography (PET).

The method may include administering a composition that includes one ormore image enhancing agents, e.g., gadolinium ions, lanthanum ions,manganese ions, iron, chromium, copper, cobalt, nickel, fluorine,dysprosium, rhenium, europium, terbium, holmium, neodymium, or mixturesthereof. As such, the method may include performing an imaging techniquesuch as magnetic resonance imaging (MRI).

The method may include administering a composition that includes one ormore radioopaque agents or contrast agents such as barium, diatrizoate,ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid,iodamide, iodipamide, iodoxamic acid, iogulamide, iohexyl, iopamidol,iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamidemeglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid,iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine,metrizamide, metrizoate, propyliodone, thallous chloride, orcombinations thereof. The method may include performing an X-ray orcomputed tomography (CT).

The method may include administering a composition that includes one ormore ultrasound contrast agents, such as dextran or a liposome (e.g., agas-filled liposome). The method may include performing an ultrasoundprocedure.

In addition to the aforementioned procedures, the method may alsoinclude performing an operative, intravascular, laparoscopic, orendoscopic procedure, before, simultaneously with, or after theimmunoconjugate or composition is administered.

The method may also include administering a second or additionalcomposition that includes a therapeutic or diagnostic agent, where thesecond or additional composition is administered before, simultaneously,or after the first composition is administered. The second or additionalcomposition may include any of the aforementioned compositions. In oneembodiment the second or additional composition includes an anti-CD74binding molecule conjugated to a therapeutic or diagnostic agent. Thetherapeutic or diagnostic agent may comprise any of the aforementioneddrugs, prodrugs, toxins, enzymes, radioisotopes, immunomodulators,cytokines, hormones, antibodies, binding molecules, oligonucleotides,chelators, cations, therapeutic nuclides, agents for photodynamictherapy, diagnostic nuclides, image enhancing agents, radioopaqueagents, and/or contrasting agents. The method may also includeperforming a PET, MRI, X-ray, CT, ultrasound, operative, intravascular,laparoscopic, or endoscopic procedure.

Also disclosed are methods of preparing the aforementioned compositions(e.g., an anti-CD74 immunoconjugate by mixing one or more amphiphiliclipids to form a carrier and contacting the carrier with an anti-CD74binding molecule, (e.g., an antibody or fragment thereof)). In oneexample, the lipid contains nucleophilic carbons (e.g., within amaleimide group), and the binding molecule contains free thiol groups(e.g., disulfides treated with a reducing agent). The method may includemixing the composition with one or more therapeutic or diagnosticagents, which may be covalently, non-covalently, or otherwise associatedwith any component of the composition.

Also disclosed is a kit that includes any of the aforementionedcompositions or the components sufficient for preparing any of theaforementioned compositions. Typically, the kit includes instructionsfor administering the compositions or, where applicable, instructionsfor preparing the compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. DNA and amino acid sequences of the murine LL1 heavy and lightchain variable regions. FIG. 1A shows DNA (SEQ ID NO:22) and amino acid(SEQ ID NO:23) sequences of LL1V_(H). FIG. 1B shows DNA (SEQ ID NO:24)and amino acid (SEQ ID NO:25) sequences of the LL1V_(k). Amino acidsequences encoded by the corresponding DNA sequences are given asone-letter codes below the nucleotide sequence. Numbering of thenucleotide sequence is on the right side. The amino acid residues in theCDR regions are shown in bold and underlined. Kabat's Ig moleculenumbering is used for amino acid residues as shown by the numberingabove the amino acid residues. The residues numbered by a letterfollowing a particular digit indicates the insertion residues defined byKabat numbering scheme. The insertion residues numbered with a letterhave the same preceding digit. For example, residues 82A, 82B and 82C inFIG. 1A are indicated as 82A, B, and C.

FIG. 2. DNA and amino acid sequences of chimeric LL1 (cLL1) heavy andlight chain variable region (see, U.S. Pat. No. 7,312,318). FIG. 2Ashows DNA (SEQ ID NO:26) and amino acid (SEQ ID NO:27) sequences of cLL1V_(H). FIG. 2B shows double-stranded DNA (SEQ ID NO:28) and amino acid(SEQ ID NO:29) sequences of cLL1V_(k). Amino acid sequences encoded bythe corresponding DNA sequences are given as one-letter codes. The aminoacid residues in the CDR regions are shown in bold and underlined. Thenumbering of nucleotides and amino acids is same as that in FIG. 1.

FIG. 3. Alignment of amino acid sequences of light and heavy chainvariable regions of a human antibody, cLL1 and hLL1. FIG. 3A shows theV_(H) amino acid sequence alignment of the human antibody RF-TS3 (SEQ IDNO:30), cLL1 (SEQ ID NO:27) and hLL1 (SEQ ID NO:33).

FIG. 3B shows the V_(k) amino acid sequence alignment of the humanantibody HF-21/28 (SEQ ID NO:31), cLL1 (SEQ ID NO:29) and hLL1 (SEQ IDNO:35). Dots indicate the residues in cLL1 that are identical to thecorresponding residues in the human antibodies. Boxed regions representthe CDR regions. Both N- and C-terminal residues (underlined) of cLL1are fixed by the staging vectors used and not compared with the humanantibodies. Kabat's Ig molecule number scheme is used as in FIG. 1.

FIG. 4. DNA and amino acid sequences of humanized LL1 (hLL1) heavy andlight chain variable regions. FIG. 4A shows the DNA (SEQ ID NO:32) andamino acid (SEQ ID NO:33) sequences of hLL1V_(H). FIG. 4B shows the DNA(SEQ ID NO:34) and amino acid (SEQ ID NO: 35) sequences of hLLI V_(k).Amino acid sequences encoded by the corresponding DNA sequences aregiven as one letter codes. The amino acid residues in the CDR regionsare shown in bold and underlined. Kabat's Ig molecule numbering schemeis used for amino acid residues as in FIG. 1.

DETAILED DESCRIPTION Definitions

The following definitions are provided to facilitate understanding ofthe disclosure herein. Where a term is not specifically defined, it isused in accordance with its plain and ordinary meaning.

As used herein, the terms “a”, “an” and “the” may refer to either thesingular or plural, unless the context otherwise makes clear that onlythe singular is meant.

As used herein, the term “about” means plus or minus ten percent (10%)of a value. For example, “about 100” would refer to any number between90 and 110.

A “binding molecule,” as used herein, is any molecule that canspecifically or selectively bind to an antigen. A binding molecule mayinclude an antibody or a fragment thereof. An anti-CD74 binding moleculeis a molecule that binds to the CD74 antigen, such as an anti-CD74antibody or fragment thereof. Other anti-CD74 binding molecules may alsoinclude multivalent molecules, multispecific molecules (e.g.,diabodies), fusion molecules, aptimers, avimers, or other naturallyoccurring or recombinantly created molecules.

An “antibody” refers to a full-length (i.e., naturally occurring orformed by normal immunoglobulin gene fragment recombinatorial processes)immunoglobulin molecule (e.g., an IgG antibody) or an immunologicallyactive (i.e., antigen-binding) portion of an immunoglobulin molecule,like an antibody fragment.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv, single domain antibodies (DABs or VHHs) andthe like, including half-molecules of IgG4 (van der Neut Kolfschoten etal. (Science 2007; 317(14 Sep.):1554-1557). Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. For example, an anti-CD74 antibody fragment binds withan epitope of CD74. The term “antibody fragment” also includes isolatedfragments consisting of the variable regions, such as the “Fv” fragmentsconsisting of the variable regions of the heavy and light chains,recombinant single chain polypeptide molecules in which light and heavychain variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains including the complementarity determining regions(CDRs) of an antibody derived from one species, preferably a rodentantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a cat or dog.

A “humanized antibody” is a recombinant protein in which the CDRs froman antibody from one species; e.g., a rodent antibody, are transferredfrom the heavy and light variable chains of the rodent antibody intohuman heavy and light variable domains. The constant domains of theantibody molecule are derived from those of a human antibody.

A “human antibody” is an antibody obtained from transgenic mice thathave been genetically engineered to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. (See, e.g., McCafferty et al., Nature 348:552-553 (1990) forthe production of human antibodies and fragments thereof in vitro, fromimmunoglobulin variable domain gene repertoires from unimmunizeddonors). In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see, e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).Human antibodies may also be generated by in vitro activated B cells.(See, U.S. Pat. Nos. 5,567,610 and 5,229,275).

An “effector” is an atom, molecule, or compound that brings about achosen result. An effector may include a therapeutic agent and/or adiagnostic agent.

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includebut are not limited to antibodies, antibody fragments, drugs, toxins,enzymes, nucleases, hormones, immunomodulators, antisenseoligonucleotides, chelators, boron compounds, photoactive agents, dyesand radioisotopes.

A “diagnostic agent” is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes, contrast agents, fluorescent compoundsor molecules and enhancing agents (e.g., paramagnetic ions). Preferably,the diagnostic agents are selected from the group consisting ofradioisotopes, enhancing agents, and fluorescent compounds. In order toload an antibody component with radioactive metals or paramagnetic ions,it may be necessary to react it with a reagent having a long tail towhich are attached a multiplicity of chelating groups for binding theions. Such a tail can be a polymer such as a polylysine, polysaccharide,or other derivatized or derivatizable chain having pendant groups towhich can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), DOTA, NOTA, NETA, porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose. Chelates are coupled to the peptide antigens usingstandard chemistries. The chelate is normally linked to the antibody bya group which enables formation of a bond to the molecule with minimalloss of immunoreactivity and minimal aggregation and/or internalcross-linking. Other, more unusual, methods and reagents for conjugatingchelates to antibodies are disclosed in U.S. Pat. No. 4,824,659.Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, used with diagnostic isotopes inthe general energy range of 60 to 4,000 keV. Some useful diagnosticnuclides may include, such as ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga,⁸⁶y, ⁸⁹Zr, ⁹⁴Tc, ^(94m)Tc, ^(99m)Tc, or ¹¹¹In. The same chelates, whencomplexed with non-radioactive metals, such as manganese, iron andgadolinium are useful for MRI, when used along with the antibodies andcarriers described herein. Macrocyclic chelates such as NOTA, DOTA, andTETA are of use with a variety of metals and radiometals, mostparticularly with radionuclides of gallium, yttrium and copper,respectively. Such metal-chelate complexes can be made very stable bytailoring the ring size to the metal of interest. Other ring-typechelates such as macrocyclic polyethers, which are of interest forstably binding nuclides, such as ²²³Ra for RAIT may be used. In certainembodiments, chelating moieties may be used to attach a PET imagingagent, such as an Al-¹⁸F complex, to a targeting molecule for use in PETanalysis (see, e.g., U.S. Pat. Nos. 7,563,433 and 7,597,876, theExamples section of each of which is incorporated herein by reference.)

An “immunoconjugate” is a conjugate of a binding molecule (e.g., anantibody) with an atom, molecule, or a higher-ordered structure (e.g.,with a carrier, a therapeutic agent, or a diagnostic agent). Thediagnostic agent can comprise a radioactive or non-radioactive label, acontrast agent (such as for magnetic resonance imaging, computedtomography or ultrasound), and the radioactive label can be a gamma-,beta-, alpha-, Auger electron-, or positron-emitting isotope. A “nakedantibody” is an antibody that is not conjugated to any other agent.

A “carrier” is an atom, molecule, or higher-ordered structure that iscapable of associating with an anti-CD74 binding molecule, such as anantibody or antibody fragment. Carriers may include molecules such aslipids or polymers (e.g., amphiphilic lipids that are capable of forminghigher-ordered structures, or carbohydrates such as dextran), orhigher-ordered structures themselves, such as micelles or nanoparticles.

As used herein, the term “antibody fusion protein” is a recombinantlyproduced antigen-binding molecule in which an antibody or antibodyfragment is linked to another protein or peptide, such as the same ordifferent antibody or antibody fragment or a DDD or AD peptide. Thefusion protein may comprise a single antibody component, a multivalentor multispecific combination of different antibody components ormultiple copies of the same antibody component. The fusion protein mayadditionally comprise an antibody or an antibody fragment and atherapeutic agent. Examples of therapeutic agents suitable for suchfusion proteins include immunomodulators and toxins. One preferred toxincomprises a ribonuclease (RNase), preferably a recombinant RNase.

A “multispecific antibody” is an antibody that can bind simultaneouslyto at least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. A “multivalent antibody” is anantibody that can bind simultaneously to at least two targets that areof the same or different structure. Valency indicates how many bindingarms or sites the antibody has to a single antigen or epitope; i.e.,monovalent, bivalent, trivalent or multivalent. The multivalency of theantibody means that it can take advantage of multiple interactions inbinding to an antigen, thus increasing the avidity of binding to theantigen. Specificity indicates how many antigens or epitopes an antibodyis able to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Multispecific, multivalent antibodiesare constructs that have more than one binding site of differentspecificity. For example, a diabody, where one binding site reacts withone antigen and the other with another antigen.

A “bispecific antibody” is an antibody that can bind simultaneously totwo targets which are of different structure. Bispecific antibodies(bsAb) and bispecific antibody fragments (bsFab) may have at least onearm that specifically binds to, for example, a B-cell, T-cell, myeloid-,plasma-, and mast-cell antigen or epitope and at least one other armthat specifically binds to a targetable conjugate that bears atherapeutic or diagnostic agent. A variety of bispecific antibodies canbe produced using molecular engineering.

A “nanoparticle” refers to a particle of size ranging from 1 to 1000 nm.Typically, a nanoparticle is biodegradable, biocompatible, and itfunctions as a carrier capable of incorporating the substance to bedelivered to a targeted cell.

Preparation of Monoclonal Antibodies

The immunoconjugates and compositions described herein may includemonoclonal antibodies. Rodent monoclonal antibodies to specific antigensmay be obtained by methods known to those skilled in the art. (See,e.g., Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al.(eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (JohnWiley & Sons 1991)).

General techniques for cloning murine immunoglobulin variable domainshave been disclosed, for example, by the publication of Orlandi et al.,Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniques for constructingchimeric antibodies are well known to those of skill in the art. As anexample, Leung et al., Hybridoma 13:469 (1994), disclose how theyproduced an LL2 chimera by combining DNA sequences encoding the V_(k)and V_(H) domains of LL2 monoclonal antibody, an anti-CD22 antibody,with respective human and IgG₁ constant region domains. This publicationalso provides the nucleotide sequences of the LL2 light and heavy chainvariable regions, V_(k) and V_(H), respectively. Techniques forproducing humanized antibodies are disclosed, for example, by Jones etal., Nature 321: 522 (1986), Riechmann et al., Nature 332: 323 (1988),Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc. Nat'lAcad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437(1992), and Singer et al., J. Immun. 150: 2844 (1993).

A chimeric antibody is a recombinant protein that contains the variabledomains including the CDRs derived from one species of animal, such as arodent antibody, while the remainder of the antibody molecule; i.e., theconstant domains, is derived from a human antibody. Accordingly, achimeric monoclonal antibody can also be humanized by replacing thesequences of the murine FR in the variable domains of the chimericantibody with one or more different human FR. Specifically, mouse CDRsare transferred from heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. As simply transferring mouse CDRs into human FRs often resultsin a reduction or even loss of antibody affinity, additionalmodification might be required in order to restore the original affinityof the murine antibody. This can be accomplished by the replacement ofone or more some human residues in the FR regions with their murinecounterparts to obtain an antibody that possesses good binding affinityto its epitope. (See, e.g., Tempest et al., Biotechnology 9:266 (1991)and Verhoeyen et al., Science 239: 1534 (1988)).

A fully human antibody, i.e., human anti-CD74 antibodies or other humanantibodies, such as anti-CD22, anti-CD19, anti-CD23, anti-CD20 oranti-CD21 antibodies for combination therapy with humanized, chimeric orhuman anti-CD74 antibodies, can be obtained from a transgenic non-humananimal. (See, e.g., Mendez et al., Nature Genetics, 15: 146-156, 1997;U.S. Pat. No. 5,633,425.) Methods for producing fully human antibodiesusing either combinatorial approaches or transgenic animals transformedwith human immunoglobulin loci are known in the art (e.g., Mancini etal., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005, Comb.Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr.Opin. Phamacol. 3:544-50; each incorporated herein by reference). Suchfully human antibodies are expected to exhibit even fewer side effectsthan chimeric or humanized antibodies and to function in vivo asessentially endogenous human antibodies. In certain embodiments, theclaimed methods and procedures may utilize human antibodies produced bysuch techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22, incorporated herein by reference). The final Fabfragments were digested with restriction endonucleases and inserted intothe bacteriophage genome to make the phage display library. Suchlibraries may be screened by standard phage display methods. The skilledartisan will realize that this technique is exemplary only and any knownmethod for making and screening human antibodies or antibody fragmentsby phage display may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods231:11-23, incorporated herein by reference) from Abgenix (Fremont,Calif.). In the XenoMouse® and similar animals, the mouse antibody geneshave been inactivated and replaced by functional human antibody genes,while the remainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

In various embodiments, the claimed methods and compositions may utilizeany of a variety of antibodies known in the art. Antibodies of use maybe commercially obtained from a number of known sources. For example, avariety of antibody secreting hybridoma lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.). A large numberof antibodies against various disease targets, including but not limitedto tumor-associated antigens, have been deposited at the ATCC and/orhave published variable region sequences and are available for use inthe claimed methods and compositions. See, e.g., U.S. Pat. Nos.7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745;6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310;6,444,206, 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;5,677,136; 5,587,459; 5,443,953; 5,525,338, the Examples section of eachof which is incorporated herein by reference. These are exemplary onlyand a wide variety of other antibodies and their hybridomas are known inthe art. The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art.

Particular antibodies that may be of use for therapy of cancer withinthe scope of the claimed methods and compositions include, but are notlimited to, LL1 (anti-CD74), LL2 and RFB4 (anti-CD22), RS7(anti-epithelial glycoprotein-1 (EGP-1)), PAM4 and KC4 (bothanti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known asCD66e), Mu-9 (anti-colon-specific antigen-p), Immu 31 (ananti-alpha-fetoprotein), TAG-72 (e.g., CC49), Tn, J591 or HuJ591(anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (an anti-carbonic anhydrase IXMAb) and hL243 (anti-HLA-DR). Such antibodies are known in the art(e.g., U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744;6,306,393; 6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702;7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567;7,300,655; 7,312,318; 7,585,491; 7,612,180; 7,642,239; and U.S. PatentApplication Publ. No. 20040202666 (now abandoned); 20050271671; and20060193865; the Examples section of each incorporated herein byreference.) Specific known antibodies of use include hPAM4 (U.S. Pat.No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164), hA19 (U.S. Pat. No.7,109,304), hIMMU31 (U.S. Pat. No. 7,300,655), hLL1 (U.S. Pat. No.7,312,318,), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S. Pat. No.7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat. No.6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. ProvisionalPatent Application 61/145,896), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections.

Production of Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab)₂, Fab′, Fab, Fv, scFv and the like. Otherantibody fragments include, but are not limited to: the F(ab′)₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab′ fragments, which can be generated by reducingdisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fab′expression expression libraries can be constructed (Huse et al., 1989,Science, 246:1274-1281) to allow rapid and easy identification ofmonoclonal Fab′ fragments with the desired specificity.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredisclosed in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raagand M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E.Bird and B. W. Walker, “Single Chain Antibody Variable Regions,”TIBTECH, Vol 9: 132-137 (1991).

An antibody fragment can be prepared by known methods, for example, asdisclosed by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 andreferences contained therein. Also, see Nisonoff et al., Arch Biochem.Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman etal., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967),and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

A single complementarity-determining region (CDR) is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. (See, e.g., Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780).

In certain embodiments, the sequences of antibodies, such as the Fcportions of antibodies, may be varied to optimize the physiologicalcharacteristics of the conjugates, such as the half-life in serum.Methods of substituting amino acid sequences in proteins are widelyknown in the art, such as by site-directed mutagenesis (e.g. Sambrook etal., Molecular Cloning, A laboratory manual, 2^(nd) Ed, 1989). Inpreferred embodiments, the variation may involve the addition or removalof one or more glycosylation sites in the Fc sequence (e.g., U.S. Pat.No. 6,254,868, the Examples section of which is incorporated herein byreference). In other preferred embodiments, specific amino acidsubstitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797).

Anti-CD74 Antibodies

The anti-CD74 binding molecules of the present immunoconjugates andcompositions may contain specific murine CDRs that have binding affinityfor the CD74 antigen. For example, the anti-CD74 binding molecules maybe humanized, chimeric or human antibodies, and they may contain theamino acids of the CDRs of a murine anti-CD74 antibody, (e.g., themurine anti-CD74 antibody, LL1). Humanized, chimeric, and humananti-CD74 antibody or fragments thereof are described in U.S. Pat. No.7,312,318, the Examples section of which is incorporated herein byreference.

Where the anti-CD74 antibody is humanized, it may contain CDRs of alight chain variable region of a murine anti-CD74 antibody (e.g., a CDR1having an amino acid sequence RSSQSLVHRNGNTYLH (SEQ ID NO:1); a CDR2having an amino acid sequence TVSNRFS (SEQ ID NO:2); and a CDR3 havingan amino acid sequence SQSSHVPPT (SEQ ID NO:3)). The humanized anti-CD74antibody or fragment may include the heavy chain variable region of thehumanized antibody, which may include CDRs of a heavy chain variableregion of a murine anti-CD74 antibody (e.g., a CDR1 having an amino acidsequence NYGVN (SEQ ID NO:4); a CDR2 having an amino acid sequenceWINPNTGEPTFDDDFKG (SEQ ID NO:5); and a CDR3 having an amino acidsequence SRGKNEAWFAY (SEQ ID NO:6)). The humanized anti-CD74 antibody orfragment thereof may include light and heavy chain variable regionsincluding complementarity-determining regions (CDRs) of murine anti-CD74(mLL1) and human antibody constant and framework (FR) region sequences,which may be substituted with at least one amino acid from thecorresponding FRs of the murine antibody. In one embodiment, thesubstituted amino acid may be selected from amino acid residue 2, 3, 4,46, 87 and 100 of the murine LL1 light chain variable region (FIG. 3B),and amino acid residues 5, 37, 38, 46, 68, 91 and 93 of the murine heavychain variable region (FIG. 3A). In another embodiment, the antibody orfragment thereof comprises a heavy chain variable region of FIG. 4A anda light chain variable region of FIG. 4B. In a further embodiment, theantibody or fragment thereof may comprise a light and heavy chainconstant region of a human antibody or a portion thereof. The antibodyor fragment may include a humanized IgG1.

Where the anti-CD74 binding molecule includes a chimeric anti-CD74antibody, the chimeric anti-CD74 antibody or fragment thereof mayinclude a light chain variable region of a murine anti-CD74 antibody(e.g., a CDR1 having an amino acid sequence RSSQSLVHRNGNTYLH (SEQ IDNO:1); a CDR2 having an amino acid sequence TVSNRFS (SEQ ID NO:2); and aCDR3 having an amino acid sequence SQSSHVPPT (SEQ ID NO:3)). In anotherembodiment, the chimeric anti-CD74 antibody or fragment thereof mayinclude a heavy chain variable region of a murine anti-CD74 antibody(e.g., a CDR1 having an amino acid sequence NYGVN (SEQ ID NO:4); a CDR2having an amino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO:5); and aCDR3 having an amino acid sequence SRGKNEAWFAY (SEQ ID NO:6)). In afurther embodiment, the chimeric anti-CD74 antibody or fragment thereofmay include the framework (FR) regions of a murine anti-CD74 antibodyand the light and heavy chain constant regions of a human antibody.Alternatively, the chimeric antibody or fragment thereof may include aheavy chain variable region of FIG. 2A and a light chain variable regionof FIG. 2B. The chimeric antibody or fragment thereof may be a chimericIgG 1 or fragment thereof.

Where the anti-CD74 binding molecule is a human anti-CD74 antibody, thehuman anti-CD74 antibody or fragment thereof may include a light chainvariable region of the human anti-CD74 antibody (e.g., a CDR1 having anamino acid sequence RSSQSLVHRNGNTYLH (SEQ ID NO:1); a CDR2 having anamino acid sequence TVSNRFS (SEQ ID NO:2); and a CDR3 having an aminoacid sequence SQSSHVPPT (SEQ ID NO:3)). In one embodiment, the humananti-CD74 antibody or fragment thereof may include a heavy chainvariable region of the human antibody which may include CDRs of a heavychain variable region of a murine anti-CD74 antibody (e.g., a CDR1having an amino acid sequence NYGVN (SEQ ID NO:4); a CDR2 having anamino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO:5); and a CDR3 havingan amino acid sequence SRGKNEAWFAY (SEQ ID NO:6)). The human antibody orfragment thereof may be a human IgG1.

Anti-CD74 antibodies of use may include murine, chimeric, humanized orhuman antibodies that contain CDRs other than the CDRs of the LL1antibody recited above, but which still block or compete for binding toCD74 with a murine, chimeric or humanized LL1 antibody. For example,such anti-CD74 antibodies may bind to the same epitope of CD74 as theLL1 antibody. Antibody binding competition experiments are well known inthe art and may be performed using any standard techniques, described inmore detail below.

Multispecific and Multivalent Antibodies

The anti-CD74 binding molecule of the present immunoconjugates andcompositions, as well as other binding molecules with differentspecificities for use in combination therapy, can also includemultispecific antibodies (comprising at least one binding site to a CD74epitope or antigen and at least one binding site to another epitope onCD74 or another antigen), and multivalent antibodies (comprisingmultiple binding sites to the same epitope or antigen), or theantibodies can be both multivalent and multispecific.

A preferred binding molecule of the present immunoconjugates orcompositions is a fusion protein, which contains two or more Fvs, Fab'sor other antigen-binding fragments of a humanized, chimeric, human ormurine anti-CD74 antibody. Another preferred antibody fusion proteincontains one or more Fvs, Fab's or other antigen-binding fragments of ahumanized, chimeric, human or murine anti-CD74 antibody and one or moreFvs, Fab's or other fragments from antibodies specific for anotherantigen that is a tumor cell marker other than CD74. For example, thenon-CD74 antigen may be expressed by the CD74-expressing cells and mayinclude a tumor marker selected from a B-cell lineage antigen, (e.g.,CD19, CD20, or CD22 for the treatment of B-cell malignancies). Thenon-CD74 antigen may also be expressed on other CD74 positive cells thatcause other types of malignancies, such as S100 in melanoma, etc.Further, the tumor cell marker may be a non-B-cell lineage antigenselected from the group consisting of HLA-DR, CD30, CD33, CD52 MUC1 andTAC. Other useful antigens may include carbonic anhydrase IX, B7,CCCL19, CCCL21, CSAp, HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5,CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5MAbs), CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38,CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70,CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154,CEACAM5, CEACAM-6, alpha-fetoprotein (AFP), VEGF (e.g. AVASTIN®,fibronectin splice variant), ED-B fibronectin (e.g., L19), EGP-1, EGP-2(e.g., 17-1A), EGF receptor (ErbB1) (e.g., ERBITUX®), ErbB2, ErbB3,Factor H, FHL-1, Flt-3, folate receptor, Ga 733, GROB, HMGB-1, hypoxiainducible factor (HIF), HM 1.24, HER-2/neu, insulin-like growth factor(ILGF), IFN-γ, IFN-α, IFN-β, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R,IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25,IP-10, IGF-1R, Ia, HM1.24, gangliosides, HCG, the HLA-DR antigen towhich L243 binds, CD66 antigens, i.e., CD66a-d or a combination thereof,MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophage migration-inhibitoryfactor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5, placental growth factor(P1GF), PSA (prostate-specific antigen), PSMA, pancreatic cancer mucin,PAM4 antigen, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le(y), mesothelin,S100, tenascin, TAC, Tn antigen, Thomas-Friedenreich antigens, tumornecrosis antigens, tumor angiogenesis antigens, TNF-α, TRAIL receptor(R1 and R2), VEGFR, RANTES, T101, as well as cancer stem cell antigens,complement factors C3, C3a, C3b, C5a, C5, and an oncogene product.

Methods for producing bispecific antibodies include engineeredrecombinant antibodies which have additional cysteine residues so thatthey crosslink more strongly than the more common immunoglobulinisotypes. (See, e.g., FitzGerald et al, Protein Eng. 10(10):1221-1225,(1997)). Another approach is to engineer recombinant fusion proteinslinking two or more different single-chain antibody or antibody fragmentsegments with the needed dual specificities. (See, e.g., Coloma et al.,Nature Biotech. 15:159-163, (1997)). A variety of bispecific antibodiescan be produced using molecular engineering. In one form, the bispecificantibody may consist of, for example, an scFv with a single binding sitefor one antigen and a Fab fragment with a single binding site for asecond antigen. In another form, the bispecific antibody may consist of,for example, an IgG with two binding sites for one antigen and two scFvwith two binding sites for a second antigen.

Diabodies, Triabodies and Tetrabodies

The immunoconjugates and compositions disclosed herein may also includefunctional bispecific single-chain antibodies (bscAb), also calleddiabodies. (See, e.g., Mack et al., Proc. Natl. Acad. Sci., 92.:7021-7025, 1995). For example, bscAb are produced by joining twosingle-chain Fv fragments via a glycine-serine linker using recombinantmethods. The V light-chain (V_(L)) and V heavy-chain (V_(H)) domains oftwo antibodies of interest are isolated using standard PCR methods. TheV_(L) and V_(H) cDNA's obtained from each hybridoma are then joined toform a single-chain fragment in a two-step fusion PCR. The first PCRstep introduces the (Gly₄-Ser₁)₃ linker (SEQ ID NO:21), and the secondstep joins the V_(L) and V_(H) amplicons. Each single chain molecule isthen cloned into a bacterial expression vector. Following amplification,one of the single-chain molecules is excised and sub-cloned into theother vector, containing the second single-chain molecule of interest.The resulting bscAb fragment is subcloned into a eukaryotic expressionvector. Functional protein expression can be obtained by transfectingthe vector into Chinese Hamster Ovary cells.

For example, a humanized, chimeric or human anti-CD74 monoclonalantibody can be used to produce antigen specific diabodies, triabodies,and tetrabodies. The monospecific diabodies, triabodies, and tetrabodiesbind selectively to targeted antigens and as the number of binding siteson the molecule increases, the affinity for the target cell increasesand a longer residence time is observed at the desired location. Fordiabodies, the two chains comprising the V_(H) polypeptide of thehumanized CD74 antibody connected to the V_(K) polypeptide of thehumanized CD74 antibody by a five amino acid residue linker may beutilized. Each chain forms one half of the humanized CD74 diabody. Inthe case of triabodies, the three chains comprising V_(H) polypeptide ofthe humanized CD74 antibody connected to the V_(K) polypeptide of thehumanized CD74 antibody by no linker may be utilized. Each chain formsone third of the hCD74 triabody.

More recently, a tetravalent tandem diabody (termed tandab) with dualspecificity has also been reported (Cochlovius et al., Cancer Research(2000) 60: 4336-4341). The bispecific tandab is a dimer of two identicalpolypeptides, each containing four variable domains of two differentantibodies (V_(H1), V_(L1), V_(H2), V_(L2)) linked in an orientation tofacilitate the formation of two potential binding sites for each of thetwo different specificities upon self-association.

Conjugated Anti-CD74 Antibodies

In various embodiments, a conjugated anti-CD74 antibody or fragmentthereof may be used to prepare an immunoconjugate or composition. Incertain embodiments, additional amino acid residues may be added toeither the N- or C-terminus of the anti-CD74 antibody or fragment. Theadditional amino acid residues may comprise a peptide tag, a signalpeptide, a cytokine, an enzyme (for example, a pro-drug activatingenzyme), a hormone, a peptide toxin, such as Pseudomonas exotoxin, apeptide drug, a cytotoxic protein or other functional proteins. As usedherein, a functional protein is a protein that has a biologicalfunction.

In another embodiment, a polymeric carrier may be conjugated to theanti-CD74 antibody or fragment thereof. See, e.g., Ryser et al., Proc.Natl. Acad. Sci. USA, 75:3867-3870, 1978, U.S. Pat. No. 4,699,784 andU.S. Pat. No. 4,046,722, which are incorporated herein by reference.Conjugation preferably does not significantly affect the bindingspecificity or affinity of the anti-CD74 antibody or fragment thereof.The carrier may be a polymer, lipid, nanoparticle, micelle or othermolecule or composite. In certain embodiments, the carrier may becovalently or non-covalently attached to one or more therapeutic and/ordiagnostic agents.

In one embodiment, drugs, toxins, radioactive compounds, enzymes,hormones, cytotoxic proteins, chelates, cytokines and other functionalagents may be conjugated to the anti-CD74 antibody or fragment,preferably through covalent attachments to the side chains of the aminoacid residues of the anti-CD74 antibody or fragment, for example amine,carboxyl, phenyl, thiol or hydroxyl groups. Various conventional linkersmay be used for this purpose, for example, diisocyanates,diisothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides,maleimide-hydroxysuccinimide esters, glutaraldehyde and the like.Conjugation of agents to the anti-CD74 antibody or fragment thereofpreferably does not significantly affect the protein's bindingspecificity or affinity to its target.

In still other embodiments, antibody-directed delivery of therapeuticsor prodrug polymers to in vivo targets can be combined with antibodydelivery of radionuclides, such that combination chemotherapy andradioimmunotherapy is achieved. Each therapeutic agent can be conjugatedto a targetable conjugate and administered simultaneously, or thenuclide can be given as part of a first targetable conjugate and thedrug given in a later step as part of a second targetable conjugate.

Dock-and-Lock (DNL)

In certain preferred embodiments, bispecific or multispecific antibodiesmay be produced using the dock-and-lock technology (see, e.g., U.S. Pat.Nos. 7,521,056; 7,550,143; 7,534,866; 7,527,787 and U.S. PatentApplication Publ. No. 20090060862; the Examples section of each of whichis incorporated herein by reference). The DNL method exploits specificprotein/protein interactions that occur between the regulatory (R)subunits of cAMP-dependent protein kinase (PKA) and the anchoring domain(AD) of A-kinase anchoring proteins (AKAPs) (Baillie et al., FEBSLetters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell. Biol.2004; 5: 959). PKA, which plays a central role in one of the beststudied signal transduction pathways triggered by the binding of cAMP tothe R subunits of cAMP-dependent protein kinase, was first isolated fromrabbit skeletal muscle in 1968 (Walsh et al., J. Biol. Chem. 1968;243:3763). The structure of the holoenzyme consists of two catalyticsubunits held in an inactive form by the R subunits (Taylor, J. Biol.Chem. 1989; 264:8443). Isozymes of PKA are found with two types of Rsubunits (R1 and RII), and each type has α and β isoforms (Scott,Pharmacol. Ther. 1991; 50:123). The R subunits have been isolated onlyas stable dimers and the dimerization domain has been shown to consistof the first 44 amino-terminal residues (Newlon et al., Nat. Struct.Biol. 1999; 6:222). Binding of cAMP to the R subunits leads to therelease of active catalytic subunits for a broad spectrum ofserine/threonine kinase activities, which are oriented toward selectedsubstrates through the compartmentalization of PKA via its docking withAKAPs (Scott et al., J. Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci. USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell. Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). Interestingly, AKAPs will only bind to dimeric Rsubunits. For human RIIα, the AD binds to a hydrophobic surface formedby the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol.1999; 6:216). Thus, the dimerization domain and AKAP binding domain ofhuman RIIα are both located within the same N-terminal 44 amino acidsequence (Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al.,EMBO J. 2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the bindinginteraction between DDD and AD as an excellent pair of linker modulesfor docking any two entities, referred to hereafter as A and B, into anoncovalent complex, which could be further locked into a stablytethered structure through the introduction of cysteine residues intoboth the DDD and AD at strategic positions to facilitate the formationof disulfide bonds. The general methodology of the “dock-and-lock”approach is as follows. Entity A is constructed by linking a DDDsequence to a precursor of A, resulting in a first component hereafterreferred to as a. Because the DDD sequence would effect the spontaneousformation of a dimer, A would thus be composed of a₂. Entity B isconstructed by linking an AD sequence to a precursor of B, resulting ina second component hereafter referred to as b. The dimeric motif of DDDcontained in a₂ will create a docking site for binding to the ADsequence contained in b, thus facilitating a ready association of a₂ andb to form a binary, trimeric complex composed of a₂b. This binding eventis made irreversible with a subsequent reaction to covalently secure thetwo entities via disulfide bridges, which occurs very efficiently basedon the principle of effective local concentration because the initialbinding interactions should bring the reactive thiol groups placed ontoboth the DDD and AD into proximity (Chimura et al., Proc. Natl. Acad.Sci. USA. 2001; 98:8480) to ligate site-specifically.

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors, virtually any protein or peptide may beincorporated into a DNL construct. However, the technique is notlimiting and other methods of conjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide, such as an antibody orfragment. However, the skilled artisan will realize that the site ofattachment of an AD or DDD moiety to an effector moiety may vary,depending on the chemical nature of the effector moiety and the part(s)of the effector moiety involved in its physiological activity.Site-specific attachment of a variety of effector moieties may beperformed using techniques known in the art, such as the use of bivalentcross-linking reagents and/or other chemical conjugation techniques.

In a preferred embodiment, the fusion proteins are assembled by the dockand lock (DNL) techniques disclosed in, e.g., Rossi E A, et al., ProcNatl Acad Sci USA 2006; 103:6841-6846; U.S. Pat. Nos. 7,521,056;7,550,143; 7,534,866; 7,527,787 and U.S. Patent Application Publ. No.20090060862; the Examples section of each of which is incorporatedherein by reference. Exemplary DDD and AD sequences that may be utilizedin the DNL method to form synthetic complexes are disclosed below.

DDD1 (SEQ ID NO: 7) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 8) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 9) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 10)CGQIEYLAKQIVDNAIQQAGC

DNL Sequence Variants

In alternative embodiments, sequence variants of the AD and/or DDDmoieties may be utilized in construction of the DNL complexes. Thestructure-function relationships of the AD and DDD domains have been thesubject of investigation. (See, e.g., Burns-Hamuro et al., 2005, ProteinSci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38; Alto etal., 2003, Proc Natl. Acad Sci USA 100:4445-50; Hundsrucker et al.,2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J 400:493-99;Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell24:397-408.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined in SEQ ID NO:7below. (See FIG. 1 of Kinderman et al., 2006, incorporated herein byreference.) The skilled artisan will realize that in designing sequencevariants of the DDD sequence, one would desirably avoid changing any ofthe underlined residues, while conservative amino acid substitutionsmight be made for residues that are less critical for dimerization andAKAP binding.

Human DDD sequence from protein kinase A (SEQ ID NO: 7)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (SEQ ID NO:9), with a binding constant for DDD of 0.4 nM. TheAKAP-IS sequence was designed as a peptide antagonist of AKAP binding toPKA. Residues in the AKAP-IS sequence where substitutions tended todecrease binding to DDD are underlined in SEQ ID NO:9.

AKAP-IS SEQUENCE (SEQ ID NO: 9) QIEYLAKQIVDNAIQQA

Similarly, Gold (2006) utilized crystallography and peptide screening todevelop a SuperAKAP-IS sequence (SEQ ID NO:11), exhibiting a five orderof magnitude higher selectivity for the RII isoform of PKA compared withthe RI isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, that increased bindingto the DDD moiety of RIIα. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for RIIα were residues 8, 11, 15, 16, 18, 19 and 20 (Gold etal., 2006). It is contemplated that in certain alternative embodiments,the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare DNL constructs. Other alternative sequences thatmight be substituted for the AKAP-IS AD sequence are shown in SEQ IDNO:12-14. Substitutions relative to the AKAP-IS sequence are underlined.It is anticipated that, as with the AKAP-IS sequence (SEQ ID NO:9), theAD moiety may also include additional N-terminal cysteine and glycineand C-terminal glycine and cysteine residues, as shown in SEQ ID NO:10.

SuperAKAP-IS (SEQ ID NO: 11) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 12) QIEYKAKQIVDHAIHQA(SEQ ID NO: 13) QIEYHAKQIVDHAIHQA (SEQ ID NO: 14) QIEYVAKQIVDHAIHQA

Stokka et al. (2006) also developed peptide competitors of AKAP bindingto PKA, shown in SEQ ID NO:15-17. The peptide antagonists weredesignated as Ht31 (SEQ ID NO:15), RIAD (SEQ ID NO:16) and PV-38 (SEQ IDNO:17). The Ht-31 peptide exhibited a greater affinity for the RIIisoform of PKA, while the RIAD and PV-38 showed higher affinity for RI.

Ht31 (SEQ ID NO: 15) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 16)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 17) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides is provided in Table 1 of Hundsrucker et al. (incorporatedherein by reference). Residues that were highly conserved among the ADdomains of different AKAP proteins are indicated below by underliningwith reference to the AKAP IS sequence (SEQ ID NO:9). The residues arethe same as observed by Alto et al. (2003), with the addition of theC-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006),incorporated herein by reference.) The sequences of peptide antagonistswith particularly high affinities for the RII DDD sequence are shown inSEQ ID NO:18-20.

AKAP-IS (SEQ ID NO: 9) QIEYLAKQIVDNAIQQA  AKAP7δ-wt-pep (SEQ ID NO: 18)PEDAELVRLSKRLVENAVLKAVQQY AKAP7δ-L304T-pep (SEQ ID NO: 19)PEDAELVRTSKRLVENAVLKAVQQY AKAP7δ-L308D-pep (SEQ ID NO: 20)PEDAELVRLSKRDVENAVLKAVQQY

Carr et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:7. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins.

(SEQ ID NO: 7) S HI Q IP P GL T ELLQGYT V EVLR Q QP P DLVEFA VE YF TR LR E A R A

The skilled artisan will realize that in general, those amino acidresidues that are highly conserved in the DDD and AD sequences fromdifferent proteins are ones that it may be preferred to remain constantin making amino acid substitutions, while residues that are less highlyconserved may be more easily varied to produce sequence variants of theAD and/or DDD sequences described herein.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Avimers

In certain embodiments, the binding moieties described herein maycomprise one or more avimer sequences. Avimers are a class of bindingproteins somewhat similar to antibodies in their affinities andspecificities for various target molecules. They were developed fromhuman extracellular receptor domains by in vitro exon shuffling andphage display. (Silverman et al., 2005, Nat. Biotechnol. 23:1493-94;Silverman et al., 2006, Nat. Biotechnol. 24:220.) The resultingmultidomain proteins may comprise multiple independent binding domains,that may exhibit improved affinity (in some cases sub-nanomolar) andspecificity compared with single-epitope binding proteins. (Id.) Invarious embodiments, avimers may be attached to, for example, DDD and/orAD sequences for use in the claimed methods and compositions. Additionaldetails concerning methods of construction and use of avimers aredisclosed, for example, in U.S. Patent Application Publication Nos.20040175756 (now abandoned), 20050048512 (now abandoned), 20050053973(now abandoned), 20050089932 and 20050221384 (now abandoned), theExamples section of each of which is incorporated herein by reference.

Phage Display

Certain embodiments of the claimed compositions and/or methods mayconcern binding peptides and/or peptide mimetics of various targetmolecules or target-binding molecules. Binding peptides may beidentified by any method known in the art, including but not limiting tothe phage display technique. Various methods of phage display andtechniques for producing diverse populations of peptides are well knownin the art. For example, U.S. Pat. Nos. 5,223,409; 5,622,699 and6,068,829 disclose methods for preparing a phage library. The phagedisplay technique involves genetically manipulating bacteriophage sothat small peptides can be expressed on their surface (Smith and Scott,1985, Science 228:1315-1317; Smith and Scott, 1993, Meth. Enzymol.21:228-257). In addition to peptides, larger protein domains such assingle-chain antibodies may also be displayed on the surface of phageparticles (Arap et al., 1998, Science 279:377-380).

Targeting amino acid sequences selective for a given organ, tissue, celltype or target molecule may be isolated by panning (Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162). In brief, a library of phage containing putativetargeting peptides is administered to an intact organism or to isolatedorgans, tissues, cell types or target molecules and samples containingbound phage are collected. Phage that bind to a target may be elutedfrom a target organ, tissue, cell type or target molecule and thenamplified by growing them in host bacteria.

In certain embodiments, the phage may be propagated in host bacteriabetween rounds of panning. Rather than being lysed by the phage, thebacteria may instead secrete multiple copies of phage that display aparticular insert. If desired, the amplified phage may be exposed to thetarget organs, tissues, cell types or target molecule again andcollected for additional rounds of panning. Multiple rounds of panningmay be performed until a population of selective or specific binders isobtained. The amino acid sequence of the peptides may be determined bysequencing the DNA corresponding to the targeting peptide insert in thephage genome. The identified targeting peptide may then be produced as asynthetic peptide by standard protein chemistry techniques (Arap et al.,1998, Smith et al., 1985).

In some embodiments, a subtraction protocol may be used to furtherreduce background phage binding. The purpose of subtraction is to removephage from the library that bind to targets other than the target ofinterest. In alternative embodiments, the phage library may beprescreened against a control cell, tissue or organ. For example,tumor-binding peptides may be identified after prescreening a libraryagainst a control normal cell line. After subtraction the library may bescreened against the molecule, cell, tissue or organ of interest. Othermethods of subtraction protocols are known and may be used in thepractice of the claimed methods, for example as disclosed in U.S. Pat.Nos. 5,840,841, 5,705,610, 5,670,312 and 5,492,807.

Aptamers

In certain embodiments, a targeting moiety of use may be an aptamer.Methods of constructing and determining the binding characteristics ofaptamers are well known in the art. For example, such techniques aredescribed in U.S. Pat. Nos. 5,582,981, 5,595,877 and 5,637,459, theExamples section of each incorporated herein by reference. Methods forpreparation and screening of aptamers that bind to particular targets ofinterest are well known, for example U.S. Pat. No. 5,475,096 and U.S.Pat. No. 5,270,163, the Examples section of each incorporated herein byreference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR₂,P(O)R, P(O)OR′, CO, or CNR₂, wherein R is H or alkyl (1-20C) and R′ isalkyl (1-20C); in addition, this group may be attached to adjacentnucleotides through O or S, Not all linkages in an oligomer need to beidentical.

Use of Humanized, Chimeric and Human Antibody for Treatment andDiagnosis

Compositions and/or immunoconjugates comprising humanized, chimeric orhuman monoclonal antibodies, i.e., anti-CD74 antibodies and otherantibodies described herein, are suitable for use in the therapeuticmethods and diagnostic methods as described herein. Accordingly, theimmunoconjugates or compositions may include naked humanized, chimericor human antibodies or may comprise antibodies that have been conjugatedto a carrier, a therapeutic agent, or a diagnostic agent. Theimmunoconjugates may be administered as a multimodal therapy. Forexample, additional therapeutic or diagnostic agents may be administeredbefore, simultaneously, or after administration of the immunoconjugateor composition.

The efficacy of the immunoconjugates may be enhanced by supplementingthe anti-CD74 binding molecules with one or more other bindingmolecules, (e.g., antibodies to antigens such as carbonic anhydrase IX,CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14,CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29,CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54,CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95,CD126, CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7,ED-B of fibronectin, Factor H, FHL-1, Flt-3, folate receptor, GROB,HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growthfactor-1 (ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R,IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25,IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4,MUC5, PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, tenascin,Le(y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigens,tumor necrosis antigens, TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR,P1GF, complement factors C3, C3a, C3b, C5a, C5, and an oncogene productor HLA-DR, preferably mature HLA-DR dimer). Preferred B-cell-associatedantigens include CD19, CD20, CD21, CD22, CD23, CD46, CD52, CD74, CD80and CD5. Preferred T-cell antigens include CD4, CD8 and CD25 (the IL-2receptor). HLA-DR antigen can be used in treatment of both B-cell andT-cell disorders. Particularly preferred B-cell antigens are CD19, CD22,CD21, CD23, CD74, CD80 and HLA-DR. Particularly preferred T-cellantigens are CD4, CD8 and CD25. CD46 is an antigen on the surface ofcancer cells that block complement-dependent lysis (CDC). Preferredmalignant melanoma associated antigens are MART-1, TRP-1, TRP-2 andgp100. Further, preferred multiple myeloma-associated antigens are MUC1and CD38.

The supplemental binding molecule may be naked or conjugated with acarrier, a therapeutic agent, or a diagnostic agent, including lipids,polyers, drugs, toxins, immunomodulators, hormones, enzymes andtherapeutic radionuclides. The supplemental binding molecule may beadministered concurrently, sequentially, or according to a prescribeddosing regimen, with the anti-CD74 immunoconjugate.

Further contemplated herein is the administration of an immunoconjugatefor diagnostic and therapeutic uses in B cell lymphomas and otherdisease or disorders. An immunoconjugate is a molecule comprising abinding molecule conjugated to a carrier. The immunoconjugate may beused to form a composition that further includes a therapeutic ordiagnostic agent, which may include a peptide that may bear thediagnostic or therapeutic agent. An immunoconjugate retains theimmunoreactivity of the binding molecule, (i.e., the antibody moiety hasabout the same or slightly reduced ability to bind the cognate antigenafter conjugation as before conjugation). Immunoconjugates may includebinding molecule conjugated to any suitable second molecule, e.g.,lipids, proteins, carbohydrates, (which may form higher-orderedstructures), or higher-ordered structures themselves, such as micellesand/or nanoparticles. It may be desirable to conjugate an anti-CD74antibody to one or more molecules that are capable of forminghigher-ordered structures (e.g., amphiphilic or amphipathic lipids).Amphiphilic molecules may also be desirable to facilitate delivery ofeffectors that demonstrate limited solubility in aqueous solution.

A wide variety of diagnostic and therapeutic reagents can be used toform the immunoconjugates and compositions as described herein.Therapeutic agents include, for example, chemotherapeutic drugs such asvinca alkaloids, anthracyclines, epidophyllotoxins, taxanes,antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors,antimitotics, antiangiogenic and apoptotic agents, particularlydoxorubicin, methotrexate, taxol, CPT-11, camptothecans, and others fromthese and other classes of anticancer agents, and the like. Other usefulcancer chemotherapeutic drugs for the preparation of immunoconjugatesand antibody fusion proteins include nitrogen mustards, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2inhibitors, pyrimidine analogs, purine analogs, platinum coordinationcomplexes, hormones, and the like. Suitable chemotherapeutic agents aredescribed in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (MackPublishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICALBASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as wellas revised editions of these publications. Other suitablechemotherapeutic agents, such as experimental drugs, are known to thoseof skill in the art.

Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA can beconjugated to one or more components of the compositions as describedherein. Alternatively, a suitable peptide including a detectable label,(e.g., a fluorescent molecule), or a cytotoxic agent, (e.g., a heavymetal or radionuclide), can be covalently, non-covalently, or otherwiseassociated with more components of the compositions as described herein.For example, a therapeutically useful immunoconjugate can be obtained byincorporating a photoactive agent or dye in the composition as describedherein. Fluorescent compositions, such as fluorochrome, and otherchromogens, or dyes, such as porphyrins sensitive to visible light, havebeen used to detect and to treat lesions by directing the suitable lightto the lesion. In therapy, this has been termed photoradiation,phototherapy, or photodynamic therapy (Joni et al. (eds.), PHOTODYNAMICTHERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van denBergh, Chem. Britain 22:430 (1986)). Moreover, monoclonal antibodieshave been coupled with photoactivated dyes for achieving phototherapy.Mew et al., J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380(1985); Oseroff et al., Proc. Natl. Acad. Sci. USA 83:8744 (1986);idem., Photochem. Photobiol. 46:83 (1987); Hasan et al., Prog. Clin.Biol. Res. 288:471 (1989); Tatsuta et al., Lasers Surg. Med. 9:422(1989); Pelegrin et al., Cancer 67:2529 (1991). Endoscopic applicationsare also contemplated. Endoscopic methods of detection and therapy aredescribed in U.S. Pat. Nos. 4,932,412; 5,525,338; 5,716,595; 5,736,119;5,922,302; 6,096,289; and 6,387,350, the Examples section of each ofwhich is incorporated herein by reference. Thus, contemplated herein isthe therapeutic use of anti-CD74 immunoconjugate compositions comprisingphotoactive agents or dyes, and the present diagnostic/therapeuticmethods may include the diagnostic or therapeutic use of anti-CD74immunoconjugate compositions comprising photoactive agents or dyes.

Also contemplated is the use of radioactive and non-radioactive agentsas diagnostic agents in the anti-CD74 immunoconjugate compositions asdescribed herein. A suitable non-radioactive diagnostic agent is acontrast agent suitable for magnetic resonance imaging, computedtomography or ultrasound. Magnetic imaging agents include, for example,non-radioactive metals, such as manganese, iron and gadolinium,complexed with metal-chelate combinations that include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, when used along with theantibodies described herein. (See U.S. Ser. No. 09/921,290 filed on Oct.10, 2001, which is incorporated in its entirety by reference).

Furthermore, the anti-CD74 immunoconjugate compositions may include aradioisotope or a positron-emitter useful for diagnostic imaging.Suitable radioisotopes may include those in the energy range of 60 to4,000 keV. Suitable radioisotopes may include ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu,⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc, ^(94m)Tc, ^(99m)Tc, ¹¹¹In, ¹²³I,¹²⁴I, ¹²⁵I, ¹³¹I, and like.

A toxin, such as Pseudomonas exotoxin, may also be present in theanti-CD74 immunoconjugate compositions as described herein. For example,the toxin may be complexed to or form the therapeutic agent portion ofan antibody fusion protein of an anti-CD74 antibody described herein.Other toxins include ricin, abrin, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. (See,e.g., Pastan et al., Cell 47:641 (1986), and Goldenberg, C A—A CancerJournal for Clinicians 44:43 (1994)). Additional toxins suitable for useherein are known to those of skill in the art and are disclosed in U.S.Pat. No. 6,077,499, the Examples section of which is incorporated hereinby reference.

An immunomodulator, such as a cytokine may also be present in theadministered anti-CD74 immunoconjugate compositions as described herein.For example, an immunomodulator may be conjugated to, or form thetherapeutic agent portion of an antibody fusion protein or beadministered as part of the anti-CD74 immunoconjugate compositions asdescribed herein. Suitable cytokines include, but are not limited to,interferons and interleukins, as described below.

Preparation of Immunoconjugates

The immunoconjugates described herein can be prepared by known methodsof linking antibodies with lipids, carbohydrates, protein, or otheratoms and molecules. For example, the binding molecules described hereincan be conjugated with one or more of the carriers described herein(e.g., lipids, polymers, micelles, or nanoparticles) to form animmunoconjugate. In certain embodiments, the immunoconjugate canincorporate a therapeutic or diagnostic agent either covalently,non-covalently. Further, any of the binding molecules described hereincan be further conjugated with one or more therapeutic or diagnosticagents described herein, or additional carriers. Generally, onetherapeutic or diagnostic agent may be attached to each binding moleculebut more than one therapeutic agent or diagnostic agent can be attachedto the same binding molecule. The antibody fusion proteins contemplatedherein comprise two or more antibodies or fragments thereof and each ofthe antibodies that comprises this fusion protein may be conjugated withone or more of the carriers described herein. Additionally, one or moreof the antibodies of the antibody fusion protein may have one or moretherapeutic of diagnostic agent attached. Further, the therapeutic donot need to be the same but can be different therapeutic agents. Forexample, the compositions described herein may include a drug and aradioisotope.

For example, an IgG can be radiolabeled with ¹³¹I and conjugated to alipid, such that the IgG-lipid conjugate can form a micelle. The micellemay incorporate one or more therapeutic or diagnostic agents, (e.g., adrug such as FUdR-dO). Alternatively, in addition to the carrier, theIgG may be conjugated to ¹³¹I (e.g., at a tyrosine residue) and a drug(e.g., at the epsilon amino group of a lysine residue), and the carriermay incorporate an additional therapeutic or diagnostic agent.Therapeutic and diagnostic agents may be covalently associated with thebinding molecule, (e.g., conjugated to reduced disulfide groups,carbohydrate side chains, or any other reactive group on the bindingmolecule. However, the skilled artisan will realize that thepolymer-conjugated, lipid-conjugated or nanoparticle-conjugatedantibodies may be used without any additional therapeutic or diagnosticagents to induce target cell death.

A carrier, therapeutic agent, or diagnostic agent can be attached at thehinge region of a reduced antibody component via disulfide bondformation. As an alternative, peptides or other molecules containingreactive moieties can be attached to an antibody component using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well known in theart. (See, e.g., Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995)). Alternatively, the carrier,therapeutic agent, or diagnostic agent can be conjugated via acarbohydrate moiety in the Fc region of an antibody. The carbohydrategroup can be used to increase the loading of the same peptide that isbound to a thiol group, or the carbohydrate moiety can be used to bind adifferent peptide.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well known to those of skill in the art. (See,e.g., Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al., Int. J.Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No. 5,057,313, all ofwhich are incorporated in their entirety by reference). Similarchemistry can be used to conjugate one or more anti-CD74 bindingmolecules to one or more carriers, therapeutic agents, or diagnosticagents. The general method involves reacting an antibody componenthaving an oxidized carbohydrate portion with a carrier polymer that hasat least one free amine function and that is loaded with a plurality ofpeptide. This reaction results in an initial Schiff base (imine)linkage, which can be stabilized by reduction to a secondary amine toform the final conjugate.

The Fc region may be absent if the anti-CD74 binding molecule is anantibody fragment. However, it is possible to introduce a carbohydratemoiety into the light chain variable region of a full-length antibody orantibody fragment. (See, e.g., Leung et al., J. Immunol. 154: 5919(1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995), Leung et al.,U.S. Pat. No. 6,254,868, the Examples section of each incorporatedherein by reference). The engineered carbohydrate moiety may be used toattach a carrier or a therapeutic or diagnostic agent.

Carriers (Lipids, Micelles, Polymers, and Nanoparticles)

The formation of micelles is known in the art. (See, e.g., Wrobel etal., Biochimica et Biophysica Acta, 1235:296 (1995); Lundberg et al., J.Pharm. Pharmacol., 51:1099-1105 (1999); Lundberg et al., Int. J. Pharm.,205:101-108 (2000); Lundberg, J. Pharm. Sci., 83:72-75 (1994); Xu etal., Molec. Cancer Ther., 1:337-346 (2002); Torchilin et al., Proc.Nat'l. Acad. Sci., 100:6039-6044 (2003). See also U.S. Pat. No.5,565,215; U.S. Pat. No. 6,379,698; and U.S. Pat. No. 6,858,226, theExamples section of each of which is incorporated herein by reference).Nanoparticles or nanocapsules formed from polymers, silica, or metals,which are useful for drug delivery or imaging, have been described aswell. (See, e.g., West et al., Applications of Nanotechnology toBiotechnology, 11:215-217 (2000); U.S. Pat. No. 5,620,708; U.S. Pat. No.5,702,727; and U.S. Pat. No. 6,530,944).

Pharmaceutically Acceptable Excipients

The immunoconjugates or compositions may include one or morepharmaceutically suitable excipients, one or more additionalingredients, or some combination of these.

The immunoconjugate or compositions disclosed herein can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby the immunoconjugate or compositions are combinedin a mixture with a pharmaceutically suitable excipient. Sterilephosphate-buffered saline is one example of a pharmaceutically suitableexcipient. Other suitable excipients are well known to those in the art.(See, e.g., Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERYSYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

The immunoconjugate or compositions disclosed herein can be formulatedfor intravenous, intramuscular or subcutaneous administration via, forexample, bolus injection or continuous infusion. Formulations forinjection can be presented in unit dosage form, e.g., in ampules or inmulti-dose containers, with an added preservative. The compositions cantake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic or diagnostic conjugate or nakedantibody. Control release preparations can be prepared through the useof polymers to complex or adsorb the immunoconjugate or naked antibody.For example, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release of animmunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate or antibody within the matrix, and the size of dispersedparticles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The immunoconjugate or compositions may also be administered to a mammalsubcutaneously or even by other parenteral routes. Moreover, theadministration may be by continuous infusion or by single or multipleboluses. In general, the dosage of an administered immunoconjugate,fusion protein or naked antibody for humans will vary depending uponsuch factors as the patient's age, weight, height, sex, general medicalcondition and previous medical history. Typically, it is desirable toprovide the recipient with a dosage of immunoconjugate or compositionincluding the immunoconjugate that is in the range of from about 1 mg/kgto 20 mg/kg as a single intravenous infusion, although a lower or higherdosage also may be administered as circumstances dictate. This dosagemay be repeated as needed, for example, once per week for 4-10 weeks,preferably once per week for 8 weeks, and more preferably, once per weekfor 4 weeks. It may also be given less frequently, such as every otherweek for several months. The dosage may be given through variousparenteral routes, with appropriate adjustment of the dose and schedule.

For purposes of therapy, the immunoconjugate, or composition includingthe immunoconjugate, is administered to a mammal in a therapeuticallyeffective amount. A suitable subject for the therapeutic and diagnosticmethods disclosed herein is usually a human, although a non-human animalsubject is also contemplated. An antibody preparation is said to beadministered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient mammal. In particular, an antibody preparationis physiologically significant if its presence invokes an antitumorresponse or mitigates the signs and symptoms of an autoimmune diseasestate. A physiologically significant effect could also be the evocationof a humoral and/or cellular immune response in the recipient mammal.

Methods of Treatment

Contemplated herein is the use of immunoconjugates or compositionsincluding immunoconjugates for treatment of a CD74 expressing disease.The disease or disorder may be selected from the group consisting ofcancer, neoplasia, an immune dysregulation disease, an autoimmunedisease, organ graft rejection, and graft versus host disease. The CD74expressing disease may be selected from the group consisting of a solidtumor, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, aB-cell disease and/or a T-cell disease. The solid tumor may be selectedfrom the group consisting of a melanoma, carcinoma and sarcoma. Thecarcinoma may be selected from the group consisting of a renalcarcinoma, lung carcinoma, intestinal carcinoma and stomach carcinoma.The B-cell disease may be selected from the group consisting of indolentforms of B-cell lymphomas, aggressive forms of B-cell lymphomas, chroniclymphatic leukemias, acute lymphatic leukemias, multiple myeloma, B-celldisorders and other diseases. In particular, the compositions describedherein are particularly useful for treatment of various autoimmune aswell as indolent forms of B-cell lymphomas, aggressive forms of B-celllymphomas, chronic lymphatic leukemias, acute lymphatic leukemias,multiple myeloma, and Waldenstrom's macroglobulinemia. For example,humanized anti-CD74 antibody components and immunoconjugates can be usedto treat both indolent and aggressive forms of non-Hodgkin's lymphoma.

More specifically, the method for treating a B-cell disease may includeadministering to a subject with a B-cell related disease, a therapeuticcomposition comprising an immunoconjugate including an anti-CD74 bindingmolecule, (e.g., a humanized, chimeric, or human anti-CD74 antibody orfragment thereof or antibody fusion protein thereof), a pharmaceuticallyacceptable carrier, and optionally a therapeutic agent, wherein theB-cell disease is a lymphoma or leukemia. More specifically, the B-celldisease may be selected from indolent forms of B-cell lymphomas,aggressive forms of B-cell lymphomas, multiple myeloma, chroniclymphatic leukemias, or acute lymphatic leukemias. The immunoconjugateor composition comprising the immunoconjugate may be administeredintravenously, intramuscularly or subcutaneously at a dose of 20-2000mg. The method may further comprise administering the immunoconjugate orcomposition before, simultaneously with, or after the administration ofat least one additional therapeutic agent or diagnostic agent used totreat the B-cell disease. The additional agent may include an additionalimmunoconjugate as described herein, including a therapeutic ordiagnostic agent. A therapeutic agent may be selected from the groupconsisting of a naked antibody, an immunomodulator, a hormone, acytotoxic agent, an enzyme, and/or an antibody conjugated to at leastone immunomodulator, radioactive label, hormone, enzyme, or cytotoxicagent, or a combination thereof. The immunomodulator preferably is acytokine and the cytotoxic agent preferably is a drug or toxin. Theantibody that is administered in combination as a naked antibody or as asupplemental immunoconjugate preferably is reactive with an antigenselected from the group consisting of carbonic anhydrase IX, CCCL19,CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55,CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126,CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7, ED-B offibronectin, Factor H, FHL-1, Flt-3, folate receptor, GROB, HMGB-1,hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-1(ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R,IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10,MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5,PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR,tenascin, Le(y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreichantigens, tumor necrosis antigens, TNF-α, TRAIL receptor (R1 and R2),VEGFR, EGFR, P1GF, complement factors C3, C3a, C3b, C5a, C5, and anoncogene product.

Also contemplated herein is the treatment of a disease comprisingadministering to a subject with a CD74 antigen-positive disease otherthan lymphoma or leukemia, a therapeutic composition that includes: (1)an immunoconjugate of an anti-CD74 binding molecule and a carrier; (2)optionally, an effector moiety; and (3) a pharmaceutically acceptableexcipient. The immunoconjugate or composition may be administeredintravenously, intramuscularly or subcutaneously at a dose of 20-5000mg. Further, the immunoconjugate may be administered before,simultaneously with, or after the administration of at least oneadditional therapeutic agent or diagnostic agent. Therapeutic agents, asdescribed above and throughout the specification, may include animmunomodulator, a hormone, a cytotoxic agent, or a binding molecule(either naked or conjugated to at least one immunomodulator, radioactivelabel, enzyme, hormone, cytotoxic agent, antisense oligonucleotide, or acombination thereof, where the immunomodulator preferably is a cytokineand the cytotoxic agent preferably is a drug or toxin). A therapeuticagent or diagnostic agent may include the compositions orimmunoconjugates as disclosed herein. When an antibody is administeredin combination with the therapeutic and/or diagnostic composition totreat a malignancy that is not a B-cell malignancy, it should bereactive with a tumor marker other than CD74, which is expressed by thecells that comprise the malignancy that is treated, and the antibodyshould be formulated in a pharmaceutically acceptable vehicle. Examplesof antibodies that can be administered for malignant melanoma associatedantigens are those antibodies reactive with MART-1, TRP-1, TRP-2 andgp100. Further, preferred antibodies to multiple myeloma-associatedantigens are those reactive with MUC1 and CD38.

The compositions for treatment contain at least one immunoconjugate,which typically includes a humanized, chimeric or human monoclonalanti-CD74 antibody alone or in combination with other antibodies, suchas other humanized, chimeric, or human antibodies. In particular,combination therapy wherein the immunoconjugate includes a fully humanantibody is also contemplated.

The compositions also may include an immunomodulator as an effector. Asused herein, the term “immunomodulator” includes may be selected from acytokine, a stem cell growth factor, a lymphotoxin, an hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN),erythropoietin, thrombopoietin and a combination thereof. Specificallyuseful are lymphotoxins such as tumor necrosis factor (TNF),hematopoietic factors, such as interleukin (IL), colony stimulatingfactor, such as granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage-colony stimulating factor (GM-CSF), interferon,such as interferons-α, -β or -γ, and stem cell growth factor, such asthat designated “S1 factor”. Included among the cytokines are growthhormones such as human growth hormone, N-methionyl human growth hormone,and bovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-B; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT. As used herein, the term cytokine includes proteins from naturalsources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines. The immunomodulator may bepresent in the composition, or alternatively, the immunomodulator can beadministered before, simultaneously with, or after administration of thetherapeutic and/or diagnostic compositions. As discussed supra, theanti-CD74 antibody may also be conjugated to the immunomodulator. Theimmunomodulator may also be conjugated to a hybrid antibody consistingof one or more antibodies binding to different antigens.

Multimodal therapies contemplated herein further include immunotherapywith immunoconjugates that include anti-CD74 binding moleculessupplemented with administration of additional binding molecules, (e.g.,anti-CD22, anti-CD19, anti-CD21, anti-CD20, anti-CD80, anti-CD23,anti-CD46 or HLA-DR, preferably the mature HLA-DR dimer antibodies inthe form of naked antibodies, fusion proteins, or as immunoconjugates).Further, a micelle or nanoparticle, as described herein, may includebinding molecules in addition to anti-CD74 binding molecules. Usefulantibodies may be polyclonal, monoclonal, chimeric, human or humanizedantibodies that recognize at least one epitope on the above-notedantigenic determinants. For example, anti-CD19 and anti-CD22 antibodiesare known to those of skill in the art. (See, e.g., Ghetie et al.,Cancer Res. 48:2610 (1988); Hekman et al., Cancer Immunol. Immunother.32:364 (1991); Longo, Curr. Opin. Oncol. 8:353 (1996) and U.S. Pat. Nos.5,798,554 and 6,187,287.)

In another form of multimodal therapy, subjects receive anti-CD74immunoconjugates, in conjunction with standard cancer chemotherapy. Forexample, “CVB” (1.5 g/m² cyclophosphamide, 200-400 mg/m² etoposide, and150-200 mg/m² carmustine) is a regimen used to treat non-Hodgkin'slymphoma. Patti et al., Eur. J. Haematol. 51: 18 (1993). Other suitablecombination chemotherapeutic regimens are well known to those of skillin the art. (See, e.g., Freedman et al., “Non-Hodgkin's Lymphomas,” inCANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et al. (eds.), pages2028-2068 (Lea & Febiger 1993)). As an illustration, first generationchemotherapeutic regimens for treatment of intermediate-gradenon-Hodgkin's lymphoma (NHL) include C-MOPP (cyclophosphamide,vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide,doxorubicin, vincristine, and prednisone). A useful second-generationchemotherapeutic regimen is m-BACOD (methotrexate, bleomycin,doxorubicin, cyclophosphamide, vincristine, dexamethasone andleucovorin), while a suitable third generation regimen is MACOP-B(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,bleomycin and leucovorin). Additional useful drugs include phenylbutyrate and bryostatin-1. In a preferred multimodal therapy, bothchemotherapeutic drugs and cytokines are co-administered with anantibody, immunoconjugate or fusion protein. The cytokines,chemotherapeutic drugs and antibody or immunoconjugate can beadministered in any order, or together.

In a preferred embodiment, NHL is treated with 4 weekly infusions of ahumanized anti-CD74 immunoconjugate (e.g., a therapeutic emulsion) at adose of 200-400 mg/m² weekly for 4 consecutive weeks or every-other week(iv over 2-8 hours), repeated as needed over next months/yrs. Alsopreferred, NHL is treated with 4 semi-monthly infusions as above, butcombined with epratuzumab (anti-CD22 humanized antibody) on the samedays, at a dose of 360 mg/m², given as an iv infusion over 1 hour,either before, during or after the anti-CD74 immunoconjugate infusion.Still preferred, NHL is treated with 4 weekly infusions of the anti-CD74immunoconjugate as above, combined with one or more injections of CD22antibody radiolabeled with a therapeutic isotope such as yttrium-90 (atdose of ⁹⁰Y between 5 and 35 mCi/meter-square) as one or more injectionsover a period of weeks or months.

In addition, a therapeutic composition as contemplated herein cancontain a mixture or hybrid molecules of monoclonal anti-CD74immunoconjugates directed to different, non-blocking CD74 epitopes.Accordingly, contemplated herein are therapeutic compositions comprisinga mixture of monoclonal anti-CD74 immunoconjugates that bind at leasttwo CD74 epitopes. Additionally, the immunoconjugates described hereinmay contain a mixture of anti-CD74 antibodies with varying CDRsequences.

As discussed supra, the immunoconjugates can be used for treating B celllymphoma and leukemia, and other B cell diseases or disorders as well asother malignancies in which affected or associated malignant cells arereactive with CD74. For example, anti-CD74 immunoconjugates can be usedto treat immune dysregulation disease and related autoimmune diseases,including Class-III autoimmune diseases such as immune-mediatedthrombocytopenias, such as acute idiopathic thrombocytopenic purpura andchronic idiopathic thrombocytopenic purpura, dermatomyositis, Sjogren'ssyndrome, multiple sclerosis, Sydenham's chorea, myasthenia gravis,systemic lupus erythematosus, lupus nephritis, rheumatic fever,polyglandular syndromes, bullous pemphigoid, diabetes mellitus,Henoch-Schonlein purpura, post-streptococcal nephritis, erythemanodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis ubiterans, primary biliary cirrhosis, Hashimoto'sthyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis and fibrosing alveolitis.

In particular, immunoconjugates including humanized, chimeric or humananti-CD74 antibodies or fragments thereof or antibody fusion proteinsthereof are administered to a subject with one or more of theseautoimmune diseases. The anti-CD74 immunoconjugates disclosed herein areparticularly useful in the method of treating autoimmune disorders,disclosed in U.S. Pat. No. 7,074,403, the Figures and Examples sectionof which is incorporated herein by reference. Preferably the anti-CD74immunoconjugate is administered intravenously, intramuscularly orsubcutaneously at a dose of 20-5000 mg. Further, the anti-CD74immunoconjugate may be administered before, during or after theadministration of at least one therapeutic agent or diagnostic agent.The therapeutic agent, as described above and throughout thespecification, may include an antibody, an immunomodulator, a hormone,an enzyme, a cytotoxic agent, an antibody conjugated to at least oneimmunomodulator, radioactive label, hormone, enzyme, or cytotoxic agent,antisense oligonucleotide or a combination thereof, where theimmunomodulator may be a cytokine and said cytotoxic agent may be a drugor toxin. The therapeutic agent may include an immunoconjugate asdescribed herein. Antibodies that may be administered in combination asa naked antibody or as a supplemental immunoconjugate include antibodiesthat react with carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp,HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23,CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45,CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM-6,alpha-fetoprotein (AFP), VEGF (e.g. AVASTIN®, fibronectin splicevariant), ED-B fibronectin (e.g., L19), EGP-1, EGP-2 (e.g., 17-1A), EGFreceptor (ErbB1) (e.g., ERBITUX®), ErbB2, ErbB3, Factor H, FHL-1, Flt-3,folate receptor, Ga 733, GROB, HMGB-1, hypoxia inducible factor (HIF),HM1.24, HER-2/neu, insulin-like growth factor (ILGF), IFN-γ, IFN-α,IFN-β, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6,IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IGF-1R, Ia, HM1.24,gangliosides, HCG, the HLA-DR antigen to which L243 binds, CD66antigens, i.e., CD66a-d or a combination thereof, MAGE, mCRP, MCP-1,MIP-1A, MIP-1B, macrophage migration-inhibitory factor (MIF), MUC1,MUC2, MUC3, MUC4, MUC5, placental growth factor (P1GF), PSA(prostate-specific antigen), PSMA, pancreatic cancer mucin, PAM4antigen, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le(y), mesothelin, S100,tenascin, TAC, Tn antigen, Thomas-Friedenreich antigens, tumor necrosisantigens, tumor angiogenesis antigens, TNF-α, TRAIL receptor (R1 andR2), VEGFR, RANTES, T101, as well as cancer stem cell antigens,complement factors C3, C3a, C3b, C5a, C5, an oncogene product and matureHLA-DR, preferably a mature HLA-DR dimer, formulated in apharmaceutically acceptable vehicle.

Method of Diagnosis

Also provided is a method of diagnosing a disease in a subject,diagnosed with or suspected of having at least one of the diseasesselected from the groups consisting of lymphoma, leukemia, myeloma,other CD74-expressing malignancies, immune dysregulation disease,autoimmune disease and a combination thereof, comprising administeringto said subject a diagnostically effective amount of a composition thatincludes (1) an immunoconjugate including at least one anti-CD74 bindingmolecule conjugated to a carrier, (2) a diagnostic agent, and (3) apharmaceutically acceptable excipient. The diagnostic agent may becovalently, non-covalently, or otherwise associated with one or morecomponents of the composition. A useful diagnostic agent may include aradioisotope, wherein the photons of the radioisotope are detected byradioscintigraphy or PET, or a metal that can be detected by MRI, or aliposome or gas filled liposome, and wherein the liposome can bedetected by an ultrasound scanning device.

The internalization of the immunoconjugate into target cells can befollowed by fluorescence labeling, essentially according to theprocedure of Pirker et al., J. Clin. Invest., 76: 1261 (1985). Further,a method for screening/diagnosing bone cancers as described in Juweid etal., 1999, could benefit from the immunoconjugates disclosed herein.Accordingly, a method comprising ^(99m)Tc-labeled humanized or chimericanti-CD74 antibody immunoconjugates is contemplated.

EXAMPLES Example 1 Preparation of Anti-CD74 Immunoliposomes CarryingFUdR-dO

Triolein (TO), egg phosphatidylcholine (EPC), dipalmitoylphosphatidylethanolamine (DPPE), cholesterol (CHOL),8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS), polyoxyethylenesorbitanmonooleate (sorbitan 80), methoxypolyethyleneglycol (mean mol. wt 2000),oleoyl chloride, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MIT) and DL-dithiotreitol (DTT) were obtained from SigmaChemical Co. (St. Louis, Mo.). Poly(ethyleneglycol)-maleimide-N-hydroxy-1-succinimidyl ester (MAL-PEG₂₀₀₀-NHS) waspurchased from Shearwater Polymers Europe (Enschede, The Netherlands).[³H]Cholesteryl oleoyl ether (COE) and [¹⁴C]dipalmitoylphosphatidylcholine were obtained from Amersham International plc(Amersham, UK). A PEG₂₀₀₀ derivative of DPPE with a maleimide group atthe distal terminus of the PEG chain (DPPE-PEG-MAL) was synthesized byreacting 25 μmol NHS-PEG-MAL with 23 μmol DPPE and 50 mmol triethylaminein chloroform for 6 h at 40° C. The product was purified by preparativesilica gel TLC. 3′,5′-O-dioleoyl-FUdR (FUdR-dO) was synthesized byadding 20 μmol oleoyl chloride and 50 μl N,N-diisopropylethylamine to 10mmol FUdR in dimethylacetamide. The mixture was incubated overnight at40° C. and then water was added to the mixture and the fatty acidderivative of FUdR was extracted with chloroform. The prodrug waspurified by preparative silica gel TLC with chloroform/methanol (95:5)as eluent.

The Burkitt's lymphoma cell lines, Raji and Ramos, Jurkat acutelymphoblastic leukemia T-cells and HL-60 myelomonocytic leukemia cells,obtained from American Type Culture Collection (Rockville, Md.), weregrown in RPMI 1640 medium with 10% heat-inactivated fetal calf serum.Cells were maintained at 37° C. and gassed with 5% CO₂ in air.

The anti-CD74 Ab, LL1, was obtained from Immunomedics, Inc. (MorrisPlains, N.J.). It was labeled with fluorescein (FITC) for quantitation.

Submicron lipid emulsions were prepared as described in detailelsewhere. (See, Lundberg, J. Pharm. Sci., 83:72-75 (1994); Lundberg etal., Int. J. Pharm., 134:119-127 (1996)). The composition of thedrug-loaded emulsions was TO, EPC, polysorbate 80, DPPE-PEG₂₀₀₀-MAL,FUdR-dO 2:2:0.8:0.6:0.3 (w/w). The components were dispensed into vialsfrom stock solutions and the solvent was evaporated to dryness underreduced pressure. Phosphate-buffered saline (PBS) was added and themixture was heated to 50° C., vortex mixed for 30 s, and sonicated witha Branson probe sonicator for 2 min.

Drug loaded liposomes were composed of EPC, DPPE-PEG₂₀₀₀-MAL, FUdR-dO1:0.2:0.1 (w/w). In experiments involving HPTS-encapsulated liposomesthe composition was EPC, CHOL, DPPE-PEG2000-MAL 2:0.5:0.4. Whenrequired, the lipid drug-carriers were labeled with trace amounts of[³H]COE. Dried lipid films were hydrated in 25 mM HEPES and 140 mM NaClbuffer (pH 7.4), (containing 35 mM HPTS when appropriate) subjected tofive freezing-thawing cycles and subsequent sonication for 2 min with aBranson probe sonicator. The phospholipid concentration was quantitatedby [¹⁴C]DPPC. MAL 2:0.5:0.4. Coupling of LL1 to lipid drug-carriers wasperformed by reaction between the maleimide (MAL) groups at the distalPEG termini on the surface of the carriers and free thiol groups on theAb. Before the coupling reaction LL1 was reduced with 50 mMdithiotreitol for 1 h at 4° C. in 0.2 M tris buffer (pH6.5). The reducedAb was separated from excess dithiotreitol by use of Sephadex G-25spin-columns, equilibrated with 50 mM sodium acetate buffered 0.9%saline (pH 5.3). The conjugation was performed in HEPES-buffered saline(pH 7.4) for 16 h at room temperature under argon. Excess maleimidegroups were blocked with 2 mM 2-mercaptoethanol for 30 min, whereafterexcess Ab and 2-mercaptoethanol were removed on a Sepharose CL-4Bcolumn. The immunoliposomes were collected near the void volume of thecolumn, passed through a 0.22 μm sterile filter and stored at 4° C. Thecoupling efficiency was estimated by use of fluorescein labeled LL1.

Example 2 Cellular Uptake and Metabolism of the Anti-CD74Immunoliposomes

Lipid drug-carriers containing the non-exchangeable marker [³H]COE wereused to study the cellular uptake of drug carrier. After completedincubation, the cells were thoroughly washed three times with cold PBSand the radioactivity measured by liquid-scintillation counting. ThepH-sensitive probe HPTS was used to study the internalization ofliposomes to low pH compartments. HPTS exhibits two major fluorescenceexcitation maxima: a peak at 403 maximal at low pH values and a peak at454 maximal at high pH values, while the fluorescence is independent ofpH at 413 nm (isobestic point). (See, Straubinger, et al., Biochemistry,29:4929-4939 (1990)). The ratio between the fluorescence at 454 nm and413 nm can be used to study the internalization of the HPTS-liposomes tointracellular acidic compartments. HPTS-liposomes were diluted to 80 μMphospholipid in HEPES buffer and added to culture dishes (4×10⁶ cells)at 37° C. After incubation for 6 h the cells were washed twice with coldPBS and the fluorescence was measured in a stirred cuvette at 20° C.Peak heights were measured at 510 nm emission at the two excitationwavelengths (413 and 454 nm) and corrected for appropriate backgroundfluorescence.

The concentration-dependent cellular association of lipid drug-carrierswith coupled LL1 and lipid drug-carriers without coupled LL1 wasexamined (not shown). Association of lipid drug-carriers with andwithout coupled LL1 was concentration dependent (not shown). TheBurkitt's lymphoma cells, Raji, showed a massive interaction withLL1-lipid drug-carrier complexes as compared to untargeted preparations(not shown). LL1-emulsion conjugates, labeled with the nontransferablecompound [³H]COE, were taken up about 50 times faster than unconjugatedemulsions by Raji cells in culture (not shown). The fast and massiveuptake of immunoemulsions is demonstrated by the fact that understandard incubation conditions about 30% of the added preparation wasassociated with cells after 24 h (not shown). The correspondingassociation of emulsions without coupled Ab was about 0.6% (not shown).The uptake values for Ramos cells were considerably lower but stillabout 30 times higher for LL1-complexes than for uncomplexed emulsions(not shown). The time-dependent association of targeted carriers wasfairly linear up to 24-h, but at prolonged incubation times the curvedeclined (not shown).

Example 3 Specificity of Immunoconjugates

The cell specificity of the preparations was tested on HL-60 and Jurkatcells. The cellular association obtained after 24 h was between 1 and 2%for both cell types with no evident difference between conjugates andplain emulsions (not shown). This extent of cellular association clearlyrepresent unspecific uptake. The specificity of the interaction wasfurther studied by measuring the cellular association of [³H]COE-labeledLL1-emulsions versus the amount of LL1 per emulsion EPC (not shown).These experiments demonstrated that association of the LL1-emulsions wasdependent on the concentration of LL1 (not shown). The specificity ofthe interaction of immunoemulsions with cells was also studied bydisplacement experiments. Free LL1 competed effectively with theLL1-emulsion complexes and at high concentrations the cellularassociation was practically abolished (not shown). These findingsstrongly indicate that LL1 preserves its immunoreactivity after bindingto lipid drug-carriers.

Example 4 Endocytosis of HPTS-Containing Immunoliposomes

The intracellular fate of LL1-liposomal complexes was studied by use ofthe pH-sensitive probe HPTS. The spectral shifts of the probe withchanges in pH make it a useful marker of the uptake and fate of theencapsulated dye. Internalization of LL1-liposomes to low-pHcompartments was demonstrated by the fluorescence ratio λ_(ex) 454/413.Values near 0.6 were obtained which corresponds to a pH value of 6.5(not shown). This value is near those obtained by other authors withHPTS-immunoliposomes. See Kirpotin, et al., Biochemistry 36 (1997)66-75; Lopes de Menezes, et al., J. Liposome Res. 9 (1999) 199-228.HPTS-liposomes without ligand gave values near 0.8, which corresponds toa pH value of about 7.0. See Lundberg, et al., Int. J. Pharm. 205 (2000)101-108. It thus seems very likely that the LL1-drug-carrier complexesare delivered to and catabolized by the lysosomes.

Example 5 Cytotoxicity Assays

Comparison of the in vitro cytotoxicity of free FUdR and FUdR-dO-loadedemulsions and liposomes with and without coupled LL1 was performed onRaji human B-cell lymphoma lines with a proliferation assay utilizingtetrazolium dye, MTT. (See, Mosmann, J. Immun. Meth., 65:55-63 (1983)).To begin, 4×10⁵ cells were plated in 24-well plates and incubated withdrug containing preparations. Control experiments included free LL1 anddrug free emulsions and liposomes. Cells were incubated for 24 h at 37°C. in an atmosphere of 95% humidity and 5% CO₂. At the 24 h time point,the cells were washed twice before replacing with fresh media andincubated for an additional 48 h. At the end of the incubation time,tetrazolium dye was added, the formed reduction product was spun down,dissolved in EtOH:DMSO 1:1 and read at 570 nm.

The cytotoxic activity of FUdR-dO in LL1 conjugated emulsions andliposomes was tested and compared with the activity of unconjugateddrug-carriers on Raji lymphoma cells (not shown). The effect of freeFUdR (in PBS) was also recorded. The cells were incubated with thevarious preparations for 24-h, followed by an additional 48-h in freshmedium. From the dose-response curves it could be seen that FUdR-dO issomewhat more efficacious in emulsions than in liposomes (not shown).However, the activity of FUdR-dO administered in both LL1-emulsions andLL1-liposomes exceeded that of FUdR (not shown). The IC70 valuesobtained were 0.45, 1.25, 5.3 and 7.3 μM for FUdR-dO loadedLL1-emulsions, LL1-liposomes, emulsions and liposomes, respectively. Thecorresponding value for FUdR was calculated to 4.35 μM. The IC₅₀ valueswere 2.5, 5.3 and 7.0 μM for LL1-emulsions, LL1-liposomes and FudR,respectively (FUdR-dO in plain emulsions and liposomes did not reachthat level (not shown).

The prodrug FUdR-dO employed in this study shows several advantageousfeatures for administration in lipid drug-carriers. It is amphiphilicand will be situated in the phospholipid monolayer and bilayer of lipidemulsions and liposomes, respectively. This makes the preparation ofdrug-carrier very convenient; the components are just mixed together andsonicated. An alternative method, which is more suited for large scaleproduction, would be the use of high pressure homogenization.

A prerequisite for site-specific delivery of the prodrug to the targetcells is the stable entrapment of the prodrug in the drug-carrier. Theunspecific transfer of FUdR-dO from carrier to cells was not actuallymeasured but the much higher cytotoxic activity of the LL1-conjugatedpreparations indicated that unspecific transfer of prodrug to cells isrelatively low (not shown). That some degree of surface transferprobably occurs finds support by a study of Koning et al., Biochim.Biophys. Acta 1420 (1999) 153-167. They found that dipalmitoyl-FUdRimmunoliposomes, without internalization, could deliver the prodrug totarget cells more efficient than liposomes without antibody.

The prodrug concept comprises a pharmacologically inactive compound thatis activated when exposed into the target cells. In this respect FUdR-dOmay fulfill the criteria of a good prodrug. It has been shown that FUdRfatty acid esters are hydrolyzed fast in cells, apparently in lysosomes.See id. An efficient intracellular liberation of the parent drug FUdR isalso indirectly supported by the high cytotoxic efficacy of the FUdR-dOpreparations.

This in vitro study demonstrates the potential for site-specificdelivery of anti-cancer drugs by use of lipid drug-carriers with LL1 astargeting ligand. Several recent studies also show that lipiddrug-carriers, even without attached ligand, can give in vivo advantageas administration vehicles for lipophilic and amphiphilic drugs. SeeConstantinides et al., Pharm. Res. 17 (2000) 175-182; Perkins et al.,Int. J. Pharm. 200 (2000) 27-39; Bom et al., J. Controlled Release 74(2001) 325-333; and Maranhao et al., Cancer. Chemother. Pharmacol. 49(2002) 487-498.

An explanation for such a favorable effect appears to be that thehalf-life of the drug increases and the tolerability is improved so thathigh doses can be administered. A recent in vivo study with nude micedemonstrated specific Ab localization of LL1 to Ramos xenografts. SeeShih et al., Cancer Immunol. Immunother. 49 (2000) 208-216. The presentstudy shows an improved cytotoxic activity of the targeted prodrugcompared to the parent drug. Immunoliposomes generally show lower orsimilar activity compared to the untargeted drug, but still demonstrateimproved efficacy in in vivo experiments. See Moase et al., Biochim.Biophys. Acta 1510 (2001) 43-55; Lopes de Menezes et al., Cancer Res. 58(1998) 3320-3330.

All patents and other references cited in the specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains, and are incorporated herein by reference,including any Tables and Figures, to the same extent as if eachreference had been incorporated by reference individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art, which are encompassed within the invention.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Also, unless indicated to the contrary, where various numerical valuesare provided for embodiments, additional embodiments are described bytaking any 2 different values as the endpoints of a range. Such rangesare also within the scope of the described invention.

What is claimed is:
 1. A method of treating a disease associated withcells that express CD74 comprising a) administering to an individualwith the disease an immunoconjugate comprising at least two anti-CD74antibodies or antigen-binding antibody fragments, wherein saidadministration results in cross-linking of CD74; and b) killing the CD74expressing cells.
 2. The method of claim 1, wherein the anti-CD74antibody or antibody fragment is selected from the group consisting of amonoclonal antibody, an antigen-binding fragment of a monoclonalantibody, a bispecific antibody, a multispecific antibody and anantibody fusion protein.
 3. The method of claim 1, wherein theimmunoconjugate comprises at least two anti-CD74 antibodies orantigen-binding fragments thereof conjugated to a complex selected fromthe group consisting of a micelle, a nanoparticle, a polymer, anemulsion, a polyethylene glycol, a lipid and a dock-and-lock (DNL)complex.
 4. The method of claim 3, wherein the immunoconjugate furthercomprises at least one therapeutic or diagnostic agent.
 5. The method ofclaim 3, wherein the anti-CD74 antibody is an LL1 antibody comprisingthe light chain variable region complementarity-determining region (CDR)sequences CDR1 (RSSQSLVHRNGNTYLH; SEQ ID NO:1), CDR2 (TVSNRFS; SEQ IDNO:2), and CDR3 (SQSSHVPPT; SEQ ID NO:3) and the heavy chain variableregion CDR sequences CDR1 (NYGVN; SEQ ID NO:4), CDR2 (WINPNTGEPTFDDDFKG;SEQ ID NO:5), and CDR3 (SRGKNEAWFAY; SEQ ID NO:6).
 6. The method ofclaim 3, wherein the anti-CD74 antibody is a chimeric, humanized orhuman antibody.
 7. The method of claim 3, wherein the anti-CD74 antibodyor fragment thereof is a naked anti-CD74 antibody or fragment thereof.8. The method of claim 7, further comprising administering at least onetherapeutic agent to said individual.
 9. The method of claim 3, whereinthe anti-CD74 antibody or fragment thereof is conjugated to at least onetherapeutic or diagnostic agent.
 10. The method of claim 3, wherein theDNL complex comprises at least one fusion protein comprising ananti-CD74 antibody or antigen-binding fragment thereof and a peptideconsisting of a dimerization and docking domain (DDD) sequence from aprotein kinase A regulatory subunit or an anchor domain (AD) sequencefrom an A-kinase anchoring protein (AKAP).
 11. The method of claim 10,wherein the DNL complex comprises a second fusion protein, wherein whenthe first fusion protein comprises a DDD sequence the second fusionprotein comprises an AD sequence, or when the first fusion proteincomprises an AD sequence then the second fusion protein comprises a DDDsequence.
 12. The method of claim 11, wherein the second fusion proteincomprises an antibody or antigen-binding fragment thereof that binds toan antigen selected from the group consisting of carbonic anhydrase IX,CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14,CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29,CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54,CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95,CD126, CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7,ED-B of fibronectin, Factor H, FHL-1, Flt-3, folate receptor, GROB,HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growthfactor-1 (ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R,IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25,IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4,MUC5, PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR,tenascin, Le(y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreichantigens, tumor necrosis antigens, TNF-α, TRAIL receptor (R1 and R2),VEGFR, EGFR, P1GF, complement factors C3, C3a, C3b, C5a, C5, and anoncogene product.
 13. The method of claim 3, wherein the anti-CD74antibodies or antibody fragments are conjugated to the complex by alinkage selected from the group consisting of a sulfide linkage, ahydrazone linkage, a hydrazine linkage, an ester linkage, an amidolinkage, an amino linkage, an imino linkage, a thiosemicarbazonelinkage, a semicarbazone linkage, an oxime linkage, a carbon-carbonlinkage, or a combination thereof.
 14. The method of claim 4, whereinthe therapeutic or diagnostic agent is selected from the groupconsisting of a drug, a prodrug, a toxin, an enzyme, a radionuclide, animmunomodulator, a cytokine, a hormone, a second antibody or antibodyfragment, an antisense oligonucleotide, an RNAi, an anti-angiogenicagent, a pro-apoptosis agent, a dye, a fluorescent agent, a contrastagent, a paramagnetic ion and a photodynamic agent.
 15. The method ofclaim 14, wherein the second antibody or antibody fragment binds to anantigen selected from the group consisting of carbonic anhydrase IX,CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14,CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29,CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54,CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95,CD126, CD133, CD138, CD147, CD154, APP, PSMA, CEACAM5, CEACAM-6, B7,ED-B of fibronectin, Factor H, FHL-1, Flt-3, folate receptor, GROB,HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growthfactor-1 (ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R,IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25,IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4,MUC5, PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR,tenascin, Le(y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreichantigens, tumor necrosis antigens, TNF-α, TRAIL receptor (R1 and R2),VEGFR, EGFR, PlGF, complement factors C3, C3a, C3b, C5a, C5, and anoncogene product.
 16. The method of claim 14, wherein the therapeuticagent is selected from the group consisting of aplidin, azaribine,anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan,calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine,celebrex, chlorambucil, cisplatin, irinotecan (CPT-11), SN-38,carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin,dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide,epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide,etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, fluorouracil,fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, leucovorin, lomustine,mechlorethamine, medroprogesterone acetate, megestrol acetate,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone,procarbazine, paclitaxel, pentostatin, PSI-341, semustine streptozocin,tamoxifen, taxanes, taxol, testosterone propionate, thalidomide,thioguanine, thiotepa, teniposide, topotecan, uracil mustard, velcade,vinblastine, vinorelbine, vincristine, ricin, abrin, ribonuclease,onconase, rapLR1, DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin.
 17. The method of claim 16, wherein thetherapeutic agent comprises FUdR, FUdR-dO, or mixtures thereof.
 18. Themethod of claim 3, wherein the immunoconjugate further comprises one ormore hard acid chelators or soft acid chelators.
 19. The method of claim18, wherein the immunoconjugate further comprises one or more cationsselected from Group II, Group III, Group IV, Group V, transition,lanthanide or actinide metal cations, or mixtures thereof, wherein saidcation is attached to the one or more hard or soft acid chelators. 20.The method of claim 19, wherein the cation is selected from the groupconsisting of Tc, Re, Bi, Cu, As, Ag, Au, At and Pb.
 21. The method ofclaim 18, wherein said chelator is selected from the group consisting ofNOTA, DOTA, DTPA, TETA, Tscg-Cys and Tsca-Cys.
 22. The method of claim14, wherein the radionuclide is selected from the group consisting of¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁶⁸Ga,⁷⁵Se, ⁷⁷As, ⁸⁶Y, ⁸⁹Sr, ⁸⁹Zr, ⁹⁰Y, ⁹⁴Tc, ^(94m)Tc, ⁹⁹Mo, ^(99m)Tc, ¹⁰⁵Pd,¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm,¹⁵⁴⁻¹⁵⁸Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra,and ²²⁵Ac.
 23. The method of claim 14, wherein the enzyme is selectedfrom the group consisting of carboxylesterase, glucuronidase,carboxypeptidase, beta-lactamase and phosphatase.
 24. The method ofclaim 14, wherein the immunomodulator is selected from the groupconsisting of a cytokine, a stem cell growth factor, a lymphotoxin, ahematopoietic factor, a colony stimulating factor (CSF), an interleukin(IL) and an interferon (IFN).
 25. The method of claim 24, wherein theimmunomodulator is selected from the group consisting of erythropoietin,thrombopoietin tumor necrosis factor-α (TNF), granulocyte-colonystimulating factor (G-CSF), granulocyte macrophage-colony stimulatingfactor (GM-CSF), interferon-α, interferon-β, interferon-γ, stem cellgrowth factor designated “S1 factor”, human growth hormone, N-methionylhuman growth hormone, bovine growth hormone, parathyroid hormone,thyroxine, insulin, proinsulin, relaxin, prorelaxin, folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), hepatic growth factor, prostaglandin,fibroblast growth factor, prolactin, placental lactogen, OB protein,mullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor, integrin, NGF-β,platelet-growth factor, TGF-α, TGF-β, insulin-like growth factor-I,insulin-like growth factor-II, macrophage-CSF (M-CSF), IL-1, IL-1 α,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3,angiostatin, thrombospondin, endostatin and LT.
 26. The method of claim14, wherein the anti-angiogenic agent is selected from the groupconsisting of angiostatin, endostatin, baculostatin, canstatin, maspin,anti-VEGF binding molecules, anti-placental growth factor bindingmolecules and anti-vascular growth factor binding molecules.
 27. Themethod of claim 3, wherein the antibody fragment is selected from thegroup consisting of F(ab′)₂, F(ab)₂, Fab, Fab′ and scFv fragments. 28.The method of claim 6, wherein the human, chimeric, or humanizedanti-CD74 antibody or fragment thereof further comprises human constantregions selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.29. The method of claim 1, wherein the disease is selected from thegroup consisting of an immune dysregulation disease, an autoimmunedisease, an organ-graft rejection, a graft-versus-host disease andcancer.
 30. The method of claim 29, wherein the cancer is selected fromthe group consisting of a solid tumor, leukemia, lymphoma, non-Hodgkin'slymphoma, Hodgkin's lymphoma, multiple myeloma, a B-cell malignancy anda T-cell malignancy.
 31. The method of claim 30, wherein the solid tumoris selected from the group consisting of a melanoma, carcinoma, sarcoma,and glioma.
 32. The method of claim 31, wherein the carcinoma isselected from the group consisting of a renal carcinoma, lung carcinoma,intestinal carcinoma, stomach carcinoma, breast carcinoma, prostatecancer and ovarian cancer.
 33. The method of claim 30, wherein theB-cell malignancy is selected from the group consisting of indolentforms of B-cell lymphomas, aggressive forms of B-cell lymphomas, chroniclymphatic leukemias, acute lymphatic leukemias and multiple myeloma 34.The method of claim 14, wherein the photodynamic agent is selected fromthe group consisting of benzoporphyrin monoacid ring A (BDP-MA), tinetiopurpurin (SnET2), sulfonated aluminum phthalocyanine (AISPc) andlutetium texaphyrin (Lutex).
 35. The method of claim 14, wherein thediagnostic agent is a radionuclide selected from the group consisting of¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc, ^(94m)Tc,^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I.
 36. The method of claim 14,wherein the diagnostic agent is a contrast agent selected from the groupconsisting of gadolinium ions, lanthanum ions, manganese ions, iron,chromium, copper, cobalt, nickel, fluorine, dysprosium, rhenium,europium, terbium, holmium, neodymium, barium, diatrizoate, ethiodizedoil, gallium citrate, iocarmic acid, iocetamic acid, iodamide,iodipamide, iodoxamic acid, iogulamide, iohexyl, iopamidol, iopanoicacid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamidemeglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid,iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine,metrizamide, metrizoate, propyliodone and thallous chloride.
 37. Themethod of claim 1, further comprising performing an operative,intravascular, laparoscopic, or endoscopic procedure.
 38. The method ofclaim 1, wherein the immunoconjugate is administered by intravenous,intramuscular, intraperitoneal, intravascular, parenteral orsubcutaneous administration.